US007732183B2

(12) United States Patent (10) Patent No.: US 7,732,183 B2 Tomasselli et al. (45) Date of Patent: *Jun. 8, 2010

(54) METHOD FOR REFOLDING ENZYMES Mallender, William; Characterization of Recombinant, Soluble B-Secretase from an Insect Cell Expression System, Molecular Phar (75) Inventors: Alfredo Tomasselli, Kalamazoo, MI macology: 59: 619-626, 2001. (US); Robert Heinrikson, Plainwell, MI Benjannet, Suzanne, et al; Post-translational Processing of f3 Secretase (3-Amyloid-converting Enzyme) and its Ectodomain (US); Donna Paddock, Kalamazoo, MI Shedding: The Journal of Biological Chemistry, vol. 276, No. 14. (US); Ana Mildner, Kalamazoo, MI Apr. 6.2001; 10879-10887. (US); Thomas Emmons, Portage, MI Capell, Anja, et al; Maturation of Pro-peptide Cleavage of (US) B-Secretase, The Journal of Biological Chemistry, vol. 275, No. 40, Oct. 6, 2000; pp. 30849-30854. (73) Assignee: Elan Pharmaceuticals, Inc., South San Charlwood, Joanne, et al; Characterization of the Blycosylation Pro Francisco, CA (US) files of Alzheimer's B-Secretase Protein Asp-2 Expressed in a Vari ety of Cell Lines; The Journal of Bioological Chemistry, vol. 276, No. (*) Notice: Subject to any disclaimer, the term of this 20, May 18, 2001; p. 16739-16748. patent is extended or adjusted under 35 Ermolieff, Jacques, et al; Proteolytic Activation of Recombinant U.S.C. 154(b) by 547 days. Pro-memapsin 2 (Pro-B-secretase) Studied with New Fluorogenic Substrates: Biochemistry 2000, 39, 12450-12456. This patent is Subject to a terminal dis Haniu, Mitsuru, et al; Chracterization of Alzheimer's B-Secretase claimer. Protein BACE. The Journal of Biological Chemistry; vol. 275, No. 28, Jul. 14, 2000: pp. 21099-21 106. (21) Appl. No.: 11/703,493 Hussain, Ishrut, etal; Identification of a Novel Aspartic Protease (Asp 2)as B Secretase, Molecular and Cellular Neuroscience; 14. 419 (22) Filed: Feb. 7, 2007 427 (1999). Khan, Amir, et al; Molecular mechanisms for the convesion of (65) Prior Publication Data zymogens to active proteolytic enzymes, Protein Science (1998) T:815-836. US 2009/0053787 A1 Feb. 26, 2009 Lin, Xinli, et al; Rearranging the domains of pepsinogen, (1995), 4:159-166. Related U.S. Application Data Lin, Xin-Li, et al; Synthesis, Purification, and Active Site Mutagenesis of Recombinant Procine Pepsinogen, The Journal of (63) Continuation of application No. 10/230,677, filed on Biological Chemistry; vol. 264, No. 8, Mar. 15, 1989: pp. 4482-4489. Aug. 29, 2002, now Pat. No. 7,186,539. Lin, Xinli, et al.; Human aspartic proteas emeapsin 2 cleaves the (60) Provisional application No. 60/316,934, filed on Aug. B -amyloid precursor protein; PNAS, Feb. 15, 2000; vol. 97, pp. 31, 2001. 1456-1460. Mildner, Ana, et al; Production of Chemokines CTAPIII and NAP/2 (51) Int. Cl. by Digestion of Recombinant Ubiquitin-CTAPIII with Yeast CI2N 9/50 (2006.01) Ubiquitin C-Terminal Hydrolase and Human Immunodeficiency Virus Protease, Protein Expression and Purification vol. 16,347-354 (52) U.S. Cl...... 435/219; 435/226 (1999). (58) Field of Classification Search ...... None Selkoe, D.J., Cell Biology of the B–Amyloid Precursor Protein and See application file for complete search history. the Genetics of alzheimer's Disease; Cold Spring Harbor Symposia (56) References Cited on Quantatative Biology, vol. LXI, 1996, pp. 587-596. Selkoe, Dennis J.; translating cell biology into therapeutic advances U.S. PATENT DOCUMENTS in Alzheimer's disease, Nature, vol. 399, Jun. 24, 199 pp. A23-A31. 5,744,346 A 4/1998 Chrysler et al. (Continued) 6,319,689 B1 1 1/2001 Powell et al. 6,323,326 B1 1 1/2001 Dorin et al. Primary Examiner Nashaat T Nashed 6,545,127 B1 4/2003 Tang et al. (74) Attorney, Agent, or Firm McDonnell Boehnen 6,583,268 B2 6, 2003 Lin Hulbert & Berghoff LLP FOREIGN PATENT DOCUMENTS (57) ABSTRACT WO 9822597 5, 1998 WO OO17369 3, 2000 WO OO47617 8, 2000 The invention provides methods for efficient recombinant WO O1OO663 1, 2001 expression, refolding, and purification of Beta-site APP WO O 123533 4/2001 cleaving enzyme (BACE) polypeptides. In various aspects, the method includes the steps of expressing a recombinant OTHER PUBLICATIONS constructin bacteria, dissolving inclusion bodies with a dena Yan, Riciang, et al.; Membrane-anchored aspartyl protease with turant at high pH in the presence of a reducing agent, diluting Alzheimer's disease B secretase activity; Nature, vol. 402, Dec. 2, the solubilized BACE polypeptide in an aqueous solutionata 1999. temperature of about 1° C. to 15° C., and incubating the Vassar, Robert, et al; B-Secretase Cleavage of Alzheimer's Amyloid diluted sample at a temperature of about 4°C. to 15°C. until Precursor Protein by the Transmembrane Aspartic Protease BACE: the recombinant BACE polypeptide folds into an active Science, vol. 286, Oct. 22, 1999. Sinha, Sukanto, et al; Purification and cloning of amyloid precursor enzyme. protein B–secretase from human brain, Nature, vol. 402, (6761):537-540. 24 Claims, 15 Drawing Sheets US 7,732,183 B2 Page 2

OTHER PUBLICATIONS Schechter, Israel, et al; On the Size of the Active Site in Proteases. I Papain, Biochemical and Biophysical Research Communications, Thinakaran, Gopal, et al; Metabolis of the "Swedish 'Amyloid Pre vol. 27, No. 2, 1967; pp. 157-162. cursor Protein Variant in Neuro 2a (N2a) Cells, the Journal of Bio Shi, Xiao-Ping, etal; The Pro Domain of B-Secretase does not Confer logical Chemistry; vol. 271, No. 16, Apr. 19, 1996; pp. 9390-9397. Strict Zymogen-like Properties but Does Assit Proper Folding of the Hong, L. et al.; Structure of the protease domain of memapsin 2 Protease Domain, vol. 276, No. 13, 2001; pp. 10366-10373. (beta-secretase) complexed with inhibitor; Science 2000; 290(5489), Heinrikson, Robert L. etal; The Biochemistry and Molecular Biology 150-3. of Recombinant Human Renin and Prorenin, Hypertension: Heinrikson, R.L., et al; The Biochemistry and molecular biology of Pathophysiology, Diagnosis and Management; 1990; pp. 1179-1196. recombinant rennin and proorenin, Hypertension: Pathophysiology, Hong, Lin, et al; Structure of the Protease Domain of Memapsin2 Diagnosis and management 1990; chapter 74:1179-1196. (B-Secretase) Complexed with Inhibitor; Science Magazine, vol. 290, Shi, X-P, et al; The pro domain of -secretase does not confer strict Oct. 6, 2000; pp. 150-153. Zymogen-like properties but assist proper refolding of the protease Selkoe, Dennis, J.; Alzheimer's Disease. Genes, Proteins and domain; J. Biol. Chem 2001; 267: 10366-10373. Therapy; Physiological Review, vol. 81, No. 2, Apr. 2001, pp. 741 Inagami, T. etal; Biomed Res. 1980; 1:456-475. T66.

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exities expresseti i r is casts: pist. .x.O., inciating treates with two protease. US 7,732,183 B2 1. 2 METHOD FOR REFOLDING ENZYMES BACE isolated from human brain is heavily glycosylated. As expressed by a stably transfected 293T cell line, BACE is CROSS-REFERENCE TO RELATED glycosylated at four asparagines: 132, 151, 202, and 333. APPLICATIONS Analysis of HEK 293 cells stably overexpressing BACE showed that the enzyme is phosphorylated at Ser477, and that This application is a continuation of U.S. patent applica phosphorylation regulates enzyme intracellular trafficking tion Ser. No. 10/230,677, filed Aug. 29, 2002, which claims (Walter et. al., 2001, J. Biol. Chem. 276:14634-41). Three the benefit of U.S. Provisional Patent Application Ser. No. disulfide bonds suggested as critical for activity, are formed 60/316,934, filed Aug. 31, 2001. between the following pairs of cysteine residues: Cys 195 10 Cys399, Cys257-Cys422, and Cys309-Cys359 (Haniu et al., FIELD OF THE INVENTION 2000, J. Biol. Chem. 275:21099-21106). These structural features of the BACE polypeptide all The invention relates generally to recombinant expression, appear to have specific functions relating to enzymatic activ refolding, and purification of an enzyme. More particularly, ity. Enzymes expressed in insect and CHO cells are properly the invention relates to novel methods for preparing purified, 15 refolded and show activity. These proteins are glycosylated. active, human BACE expressed in E. coli. For example, insect cells express glycosylated BACE, from the mannose-rich glycans available in the insect cells. Bian BACKGROUND OF THE INVENTION tennary and triantennary oligosaccharides of the complex type provide glycosylation in the CHO-expressed BACE Neuritic plaques containing primarily amyloid beta protein (Charlwoodet. al., 2001 J. Biol. Chem. 276:16739-48). These (Abeta) are one of the hallmarks of Alzheimer's Disease. glycosylated proteins have proven difficult to process, how Beta-site APP cleaving enzyme (BACE), known also as beta ever, due to the heterogeneity conferred by differential gly secretase, Asp2, and Memapsin, has been identified as the cosylation. BACE production in these cells generally requires enzyme responsible for processing amyloid precursorprotein expensive culture media and does not yield high amounts of (APP) to produce the N-terminal portion of the Abeta peptide. 25 protein. This enzyme has been suggested as rate limiting in the pro These negative aspects of mammalian and insect cell duction of the Abeta peptide. See, for example, Sinha et al., expression do not apply to BACE proteins expressed in E. 1999, Nature 402:537-554, and published PCT applications coli. Bacteria are easy to grow, produce highyields of protein, WO 00/17369, WO 01/23533, and WO 98/22597. See also: and the cost of culture media is low. However, E. coli do not Hussain, I. et al., 1999, Mol. Cell. Neurosci. 14:419-427; 30 provide for post-translational modifications of the protein. Vassar, R. et al., 1999, Science 286:735-741; Yan, R. et al., Previous attempts to produce and isolate large quantities of 2000, Nature 402:533-537; and Lin, X. et al., 2000, Proc. active BACE from E. coli were initially unsuccessful. Natl. Acad. Sci. USA 97:1456-1460 (2000). Although the cells grew well and expressed reasonable quan BACE is a therapeutic target for the development of inhibi tities of protein, refolding and isolating the enzyme using tory compounds for the treatment of Alzheimer's Disease. 35 known published methods, including those described in Lin Rational drug design methods require Supply of properly et al. 2000, PNAS USA 97:1456-60 and Tang, WO 01/00663, expressed, refolded, and active BACE in order to model and failed to produce quantities of active BACE suitable for drug design appropriate new drugs. BACE in Sufficient amounts discovery methods. has proven difficult to obtain. Accordingly, a simple, efficient, and reliable method for BACE is a relatively large and structurally complex 40 expression of recombinant BACE in E. coli and for refolding enzyme. The primary structure of BACE as it is synthesized in and purification of sufficient quantities of BACE necessary the endoplasmic reticulum is shown in FIG. 1 SEQ ID for use in drug discovery methods is greatly needed. NO:1. The enzyme contains 501 amino acids, including a N-terminal signal (leader) sequence of about 21 amino acids SUMMARY OF THE INVENTION (pre-sequence domain) followed by a pro-sequence domain 45 consisting approximately of residues 22 to 45 (pro-sequence The invention provides for a method of refolding recom domain) that is proteolytically removed once the enzyme binant BACE polypeptide. In one aspect of the invention, the reaches its destination in the Golgi apparatus, to generate a method includes: mature enzyme. a) Solubilizing a recombinant BACE polypeptide inadena Prosequence domains are commonly found in protease 50 turant at a pH of about 10-11 and in the presence of a precursor polypeptides, where they generally function to pre reducing agent, vent catalytic activity and assist in protein folding. The pro b) diluting the solubilized BACE polypeptide in an aque sequence domain is typically cleaved from the protease pre ous solution having a temperature of about 1° C. to 15° cursor to generate a mature active protease. Previous work in C., to obtain a diluted sample; and abaculovirus expression system, expressing a BACE precur 55 c) incubating the diluted sample at a temperature of about Sor having BACE pre-sequence and pro-sequence domains 4°C. to 15°C. until the recombinant BACE polypeptide but truncated at the junction between the putative protease folds into an active enzyme. and transmembrane region, indicated that the pro-sequence In various aspects of the invention, the solubilized BACE is domain facilitates proper folding of the BACE protease diluted to a final concentration of about 1 microgram/ml to domain. See Shi, X-P. et al., Mar. 30, 2001, J. of Biol. Chem. 60 about 300 micrograms/ml. Also, prior to dilution, the recom 276(13): 10366-10373. binant BACE has an absorbance at 280 nm of about 0.1 to BACE contains a transmembrane domain of about 27 about 4.0, and the solubilized BACE is diluted from about 10 amino acids that anchors the protein to the membrane. A short fold to about 150 fold. Further, the active enzyme has at least cytosolic C-terminal tail of 21 amino acids follows the trans about 40% of the activity of recombinant BACE expressed in membrane domain. Attachment to the membrane allows 65 CHO cells. BACE to interact with and cleave APP, the first and prereq In another aspect of the invention, the recombinant BACE uisite step in the generation of A-beta. is pGE70-BACE SEQID NO:3), pCRE80L-BACE SEQID US 7,732,183 B2 3 4 NO:7), orpET11a BACE SEQID NO:5 and the incubation DESCRIPTION OF THE FIGURES is from about 3 days to about 6 weeks. In yet another aspect of the invention the recombinant FIG. 1 is the polypeptide sequence of human BACE SEQ BACE is lacking all or a portion of the BACE prosequence. ID NO: 1 An additional aspect of the invention further provides for a FIGS. 2A-2C show the DNA SEQ ID NO: 4) and pre method of producing active recombinant BACE polypeptide dicted amino acid SEQ ID NO: 5 sequences of plT11a comprising: BACE a) expressing a polynucleotide encoding BACE polypep FIGS. 3A-3C show the DNA SEQ ID NO: 6) and pre tide in E. coli to produce inclusion bodies of recombi dicted amino acid SEQ ID NO: 7 sequences of pGE80L nant BACE polypeptide; 10 BACE b) solubilizing the inclusion bodies to release the BACE FIGS. 4A and 4B show the DNA SEQ ID NO: 2 and polypeptide; predicted amino acid SEQID NO: 3 sequences of pGE70 c) reducing the released BACE polypeptide with a reduc BACE ing agent, FIG. 5 is a series of graphs showing the activity of recom d) diluting the reduced BACE with an aqueous solution 15 binant BACE prepared from a series of dilutions and incu having a temperature of about 1° C. to 15° C.; and bated for various times. Initial protein concentrations in 7.5M e) incubating the diluted BACE at a temperature of about 4 urea were measured at 280 nm and adjusted to an absorbance C. to 15° C. and at a pH of about 10-11, to obtain active of 0.5 AU (a & d), 1.5 AU (b & e), and 3.0 AU (c & f) prior to BACE polypeptide. dilution with cold water. Dilution factors were 20 (squares), In this aspect of the invention, the BACE may be diluted to 35 (triangles), and 50 (circles). a final concentration of about 1 microgram/ml to about 300 FIG. 6 shows photographs of gels showing the progression micrograms/ml. The solubilized BACE, prior to dilution, may of BACE polypeptide purification from expressed inclusion have an absorbance at 280 nm of about 0.1 to about 4.0, and bodies through chromatographic purification columns. Lane the solubilized BACE may be diluted from about 10 fold to A2 shows isolated inclusion body protein; lane A4 shows the about 150 fold. The active enzyme may have at least about 25 refolded protein purified by Q-sepharose; and lane A7 shows 40% of the activity of recombinant BACE expressed in CHO the protein product after I-1 affinity chromatography. Lane cells. The diluted sample may be incubated from about 3 days B2 shows isolated inclusion body protein; lane B3 shows to about 6 weeks. The polynucleotide sequence may encode refolded protein purified through Q-sepharose; lane B4 pOE70-BACE SEQ ID NO:3), pGE80L-BACE SEQ ID through Sephacryl-200; and lane B5 shows the protein prod NO:7), or pET11a-BACE SEQ ID NO:5). The polynucle 30 uct after I-1 affinity chromatography. otide may encode a BACE lacking all or a portion of its FIG. 7 shows photographs of gels showing the progression prosequence. of BACE polypeptide purification from expressed inclusion Another aspect of the invention relates to a method for bodies through chromatographic purification columns. Lanes refolding BACE polypeptide comprising: 1 and 3, Standards. Lane 2, insoluble fraction. Lane 4, post Q a) diluting solubulized, reduced BACE polypeptide at least 35 column. Lane 5, post Q column, after the sample was dia 20 fold with a low ionic strength aqueous solution hav lyzed. Lane 6, sample of lane 5 was adjusted to pH 5.7. Lane ing a temperature of about 1 to 15° C.; 7, affinity feed. Lane 8, affinity flow through. Lane 9, wash. b) incubating the diluted BACE polypeptide at a starting Lane 10, affinity elution. pH of about 10 to 11, and at a temperature of about 1 to FIG. 8 is a photograph of a gel showing the results of 15° C. from about 2 days to about 6 weeks, and 40 SDS-PAGE analysis of recombinant BACE polypeptide pro c) recovering active, refolded BACE polypeptide. duced in E. coli. Lanes 3-6 contained protein expressed from In this aspect of the invention, the method may include the B2 construct SEQ ID NO:6), and lanes 7-10 contain adding a reducing agent to the expressed recombinant BACE protein expressed from B1 SEQ ID NO:4). Lanes 3 and 7 polypeptide prior to the diluting step. The reducing agent may show total protein obtained from uninduced cells; lanes 4 and 45 8 show total protein from induced cells; lanes 5 and 9 show be beta-mercaptoethanol. soluble protein fractions; and lanes 6 and 10 show insoluble In another aspect, the invention relates to a method for protein fractions. producing active, recombinant BACE polypeptide. The FIG.9 shows the amino acid sequence SEQID NO:23 of method includes: recombinant BACE expressed in CHO cells. a) expressing a polynucleotide encoding BACE polypep 50 FIG. 10 is shows photographs of gels showing the progres tide in E. coli. sion of BACE polypeptide purification from inclusion bodies b) isolating and purifying the expressed BACE polypeptide through various chromatographic columns, and also showing from the E. coli. treatment of purified polypeptide with HIV-1 protease. Lane c) refolding the purified BACE polypeptide by a method A2 shows inclusion bodies; lane A4 shows post Q-Sepharose that comprises diluting the purified BACE polypeptide 55 protein; lane A5 shows protein pre affinity chromatography; 20-50 fold with water having a temperature of about 1 to lane A6 shows the affinity column flow through; and lane A7 about 15° C. under conditions of reduced protein; and shows the protein product after I-1 affinity chromatography. d) recovering active recombinant BACE polypeptide. Lane B2 shows inclusion bodies; lane B4 shows protein post In another aspect, the invention relates to cleaving the Q-Sepharose; lane B6 shows protein treated with HIV-pro refolded BACE polypeptide in the presence of HIV-protease. 60 tease; lane B9 shows protein product after I-1 affinity chro In another aspect, the invention relates to expression con matography. Lane C2 shows inclusion bodies; lane C4 shows struct for the expression of recombinant BACE comprising protein post-Q-Sepharose and Sepacryl-200 purification; lane the structure of any of the constructs B1-B6. C6 shows post first I-1 affinity chromatography: lanes C7 and In another aspect, the invention relates to a fusion protein C9 shows post-dialysis; lane C10 shows after adjustment to for the expression of recombinant BACE including a poly 65 pH 5.7; lane C11 shows protein after HIV protease treatment; nucleotide encoding a BACE polypeptide and a Caspase3 lane C14 shows the protein product after second I-1 affinity cleavage site. chromatography. US 7,732,183 B2 5 6 DETAILED DESCRIPTION OF THE INVENTION Useful constructs for the production of BACE are designed to express a selected portion of the BACE polypeptide, for “BACE’ (beta-site APP-cleaving enzyme), refers to an example, express a portion of the amino acid sequence shown enzyme that mediates cleavage at the beta-site of APP. This in FIG. 1 SEQID NO:1. The polynucleotide encoding the enzyme is also known as beta-secretase, Asp2, and Memapsin 5 BACE polypeptide can be operably linked to suitable tran 2. BACE has been described, for example, in published PCT Scriptional or translational regulatory sequences in an expres patent applications: WO 00/17369, WO 00/47618 and WO sion construct. Regulatory sequences include transcriptional 01/23533, each of which is incorporated herein by reference promoters, operators, enhancers, mRNA ribosomal binding in their entirety. BACE comprises an aspartyl protease and sites, and other sequences that control transcription or trans contains the classical consensus aspartyl protease active site 10 lation. Nucleotide sequences are “operably linked when the motif (DTG/DSG). As used herein, BACE refers to the full regulatory sequence functionally relates to the polynucle length BACE or an active fragment. otide encoding BACE. Thus, a promoter nucleotide sequence Features of the BACE polypeptide shown in FIG. 1 (SEQ is operably linked to a polynucleotide encoding BACE if the ID NO. 1 include a 21 amino acid leader (signal or pre-) promoter nucleotide sequence directs the transcription of the sequence shown in italics, and a 24 amino acid pro-sequence, 15 BACE sequence. shown in boldtype. T' marks the start of the pro-sequence. A The polynucleotide is cloned into appropriate expression furin cleavage site at amino acid E is indicated by Sug vectors for expression in the appropriate host. Generally, an gesting Ef as the start of the catalytic or protease domain. expression vector will include a selectable marker and an FV indicates an HIV-1 protease cleavage site discovered at origin of replication, such as for propagation in E. coli. V', as disclosed in the Examples below. Four glycosylated Expression vectors generally comprise one or more pheno asparagines at amino acid positions 132, 151, 202, and 333 typic selectable marker genes. Such genes generally encode, are shown in bold-italics (N). A 27 amino acid transmem for example, a protein that confers antibiotic resistance or that brane domain is underlined, and is followed by the cytosolic Supplies an auxotrophic requirement. C-terminal tail. Disulphide bridges are formed by cysteines A polynucleotide can encode a BACE polypeptide having (Cys'95-Cys, Cys?57-Cys?: and Cys-Cyss) 25 an N-terminal methionine to facilitate expression of the The 24 amino acid pro-segment shown in FIG. 1 represents recombinant polypeptide in a prokaryotic host, for example, the commonly understood prosegment found in BACE puri for expression in E. coli. The N-terminal methionine can fied from human brain tissue. However, as used herein, the optionally be cleaved from the expressed BACE polypeptide. prosegment of BACE may include a greater number of amino The polynucleotide can also encode other N-terminal amino acids. As shown below, it has been discovered that BACE 30 acids added to the BACE polypeptide that facilitate expres constructs lacking amino acids up to Argor Val" provide sion in E. coli. Such amino acids include, but are not limited for an active enzyme. In addition, a sequence alignment of to, a T7 leader sequence, a T7-caspase 8 leader sequence, and BACE with other common aspartyl proteases, such as pepsi known tags for purification such as the T7-Tag MASMTG nogen and progastricsin, Suggests that the prosegment may be GQQMGRSEQID NO:8 that allows binding of antibodies, as long as about 45 amino acids. Accordingly, for the pur or a six-histidine tag (His) that allows purification by binding poses herein, the prosegment may include any number of to nickel. Other useful peptide tags include the thioredoxin amino acids as long as the amino acid sequence remaining tag, hemaglutinin tag, and GST tag. These and other amino after the deletion can be an active enzyme. acid tags can be encoded by polynucleotides added to either terminus of the polynucleotide encoding BACE. In addition, Recombinant BACE can be produced, for example, in E. 40 coli or other suitable host cells, by expressing a construct that the wild-type polynucleotide sequence expressing human contains at least a portion of a cDNA encoding BACE, for BACE can be mutated to provide for codons preferred for the example, encoding at least a portion of the amino acid expression of BACE in E. coli or other desirable host. sequence shown in FIG. 1 SEQID NO:1. The construct can The polynucleotide of the expression construct can encode also contain additional nucleotide sequences that may, for a BACE polypeptide that is truncated by removal of all or a example, assist in purification and/or expression of the 45 portion of the cytoplasmic tail, the prosequence, the trans recombinant polypeptide, as desired. membrane domain, the membrane proximal region, or any When expressed in E. coli, recombinant BACE accumu combination of these. The expression constructs can also lates intracellularly in an insoluble form, resulting in phase encode cleavage sites for selected enzymes, to improve puri bright inclusions in the cytoplasm (inclusion bodies). The 50 fication of the expressed protein or to assist in expression of protein in the inclusion bodies can be a mixture of monomeric the enzyme, when desired. and multimeric forms of the protein, both reduced and oxi It has been found that active recombinant BACE protein dized. can be expressed from constructs encoding protein having an Processes designed to recover biologically active, soluble N-terminal amino acid within the protease domain; that is, the protein from the insoluble cellular material generally include 55 amino acid sequence of the BACE protein can begin at posi the steps of: (1) cell lysis, (2) isolation of inclusion bodies, (3) tions downstream of T', for example at Rand F. In addi solubilization of protein from inclusion bodies, (4) refolding tion, as shown in FIG. 1 SEQ ID NO:1, expressed BACE of solubilized protein, and (5) purification of the active pro protein can terminate at S'', lacking the transmembrane tein. Each of these steps will be described in relation to the domain and cytosolic tail region. This provides BACE in a invention below. 60 soluble form, that is, a form that is not membrane-bound. For efficient expression, one or more codons of the poly Cloning and Expression of BACE nucleotide sequence encoding BACE can be modified, using Expression constructs and methods have been developed Such techniques as site directed mutagenesis, to eliminate for the efficient production, refolding, and purification of GC-rich regions of strong secondary structure known to inter recombinant protein, for example, recombinant human 65 fere with efficient cloning or expression of the recombinant BACE. Production, refolding, and purification of protein can protein. Codons can also be optimized for expression in E. be of protein produced in bacterial hosts, such as E. coli. coli, for example, according to published codon preferences. US 7,732,183 B2 7 8 Examples of suitable constructs for expression in E. coli Production of BACE in E. coli are presented in Table 1, where T' denotes the first amino acid An expression construct containing a polynucleotide of the prosequence, and amino acids are numbered accord encoding BACE can be used to transform bacteria, for ingly. It will be understood that modifications to the specific example E. coli, in order to produce BACE protein. Produc constructs identified herein can be made within the scope of 5 tion of the protein can be inducible or constitutive, depending the invention. upon the control elements provided in the vectors. For example, expression constructs are transfected into a bacte TABLE 1. rial host, such as E. coli BL21 codon plus (DE3) RP (Strat agene) and grown in Suitable media, such as Luria broth Expression Constructs Encoding BACE, Suitable for 10 supplemented with 100 micrograms/ml ampicillin and 34 Expression in E. coli Construct micrograms/ml chloromphenicol. When cells have grown to a Number (Vector - Encoded polypeptide) desired density, in general, when the absorbance of the culture at 550 nm is between 0.5 and 0.6, expression is induced. For B1 pET11a-T7..Tag-Gly-Ser-Met-(AGV... QTDES'') example, in the expression vectors listed in Table 1, the T7 or SEQ ID NO: 5 B2 pOE8OL-Met-Arg-Gly-Ser-(His)-Gly-Ser-Ile-Glu-Thr-Asp 15 T5 lac promoter promotes expression of the operably linked (TQH... QTDES''') SEQID NO: 7 BACE polynucleotide upon addition of IPTG (for example, to B4 pOE70-(M)RGSFVE... QTDES-2 RS(His) a final concentration of about 1 mM) to the culture media. SEQ ID NO:3) B5 pET11a-T7..Tag-Gly-Ser-Met After induction, for example, about three hours, the cell pellet (AGV ... R168(A25)L 9... QTDES-32) is collected and can be stored, generally at -70° C., for later SEQ ID NO:25) inclusion body isolation, enzyme refolding and purification. B6 pOET11a-T7..Tag-Gly-Ser-Met The expressed recombinant enzyme accumulates intracel (AGV ... Ll2(A44)P 169... QTDES-32) lularly in an insoluble form, as inclusion bodies. To recover SEQ ID NO: 27) the enzyme from insoluble cellular material, bacterial cells are pelleted from the bacterial cell culture, lysed, and the In Table 1, the constructs are numbered (B1, B2, B4, B5, 25 inclusion bodies are isolated from the lysed cells. The recom and B6) for convenience. Shown in the table are the vector binant enzyme can then be isolated from the isolated inclu name, for example, pFT23a, pCRE70, pGE80L, and PET11a. sion bodies. pET-derived vectors (pET23a and pET11a) are commercially Generally, lysing of cells to obtain the protein inclusion available from Novagen, Inc., Madison, Wis.; pGE70 and bodies can be accomplished using a number of known meth pOE80L are commercially available from Qiagen, Inc., 30 ods, including mechanical and chemical techniques. Sonica Valencia, Calif. tion and freeze-thaw techniques are generally not practical These vectors all contain the inducible lac promoter. Exog for the volume of cells being disrupted. However, any com enous sequences encoding amino acid tags, leaders, and the mercially available device that uses a pressure differential to like, are inserted upstream of the nucleic acid sequence disrupt the cells, such as a French Pressora Rannie apparatus, encoding the BACE polypeptide of the construct. The table 35 is acceptable, assuming the overall handling capacity is simi lar or greater than these instruments. Detergent solubilization illustrates the expression constructs according to the amino is not generally a practical Solution, since removal of the acid sequence encoded by the nucleic acids of the construct. detergent can pose a difficult challenge and may influence Construct B1 encodes a BACE polypeptide that begins Subsequent refolding efforts. Detergents may solubilize con within the leader sequence, at Ala (SEQ ID NO:5). Con 40 taminating proteins and nucleic acids together with some or struct B2 encodes a BACE polypeptide that begins at T SEQ all of the protein of interest from the inclusion bodies, and ID NO:7). Each of B1 and B2 encode a BACE polypeptide thus is not a desirable option. Once the cells have been lysed, lacking a transmembrane domain and cytoplasmic tail. The the inclusion bodies may be washed to remove protein con DNA and predicted amino acid sequences of these constructs taminants associated with or entrapped in the inclusion bod is shown in FIGS. 2A-2CSEQID NO:4SEQID NO:5 and 45 1CS 3A-3C SEQID NO:6 SEQID NO:7). For example, to obtain inclusion bodies, bacterial cells can Construct B4 encodes a BACE polypeptide lacking the be suspended in a Suitable buffer that may contain a salt Such BACE prosequence as well as a portion of the N-terminal as Sodium chloride, a chelating agent Such as EDTA, or both. region of the protease domain SEQ ID NO:3. In this con Suspended cells are then lysed using, for example, a French struct, the BACE polynucleotide insert encodes amino acids 50 Press or a Rannie apparatus. The insoluble cellular material R through S''. This construct does not encode a prose obtained is washed in buffer and can be stored and frozen at quence, but rather each encodes a protein that begins within -20° C. the protease domain of BACE. As shown in the Examples Protein aggregates (inclusion bodies) are solubilized and below, it has Surprisingly been found that the prosequence is then refolded to obtain active protein. Reagents that can be not required for proper refolding of expressed recombinant 55 used to solubilize BACE include denaturants such as urea, active BACE. The DNASEQ ID NO:2 and predicted amino guanidine HCl, guanidine thiocyanate, and the like, generally acid sequence SEQID NO:3 of this construct is shown in at a concentration of about 6M to 8M. Reducing agents, such FIGS. 4A and 4B. as beta-mercaptoethanol (BME), glutathione (gamma-Glu Constructs B5 and B6 each encode a BACE polypeptide Cys-Gly; or GSH, Sigma Cat. No. G-6529); or DTT (dithio having amino acid deletions within the protease domain of 60 threitol, Sigma Cat. No. D-0632), and the like can also be BACE. B5 contains a polynucleotide insert of BACE cDNA used. These reducing agents can be used separately or in obtained from brain, encoding a protein that lacks 25 amino combination to provide the isolated protein in a reduced form acids between Rand L' SEQID NO:25). B6 contains a (random coil). The reducing agents can reduce the presence polynucleotide insert of BACE cDNA obtained from pan of dimers and higher molecular weight multimers, as well as creas, encoding a protein that lacks 44 amino acids between 65 reduce improper folding, for example, as a result of cysteine L'' and P' (SEQ ID NO:27). Each of these constructs residues within the protein, or reduce aggregation of the pro expresses BACE polypeptide in E. coli. tein. US 7,732,183 B2 10 Solubilization of BACE present in inclusion bodies can be tration of recombinant BACE capable of refolding to an achieved via treatment with a solubilizing agent (denaturant) active enzyme upon incubation in a cold room from a few at a high pH (about pH 10-11), and in the presence of a days to several weeks. reducing agent such as BME. For example, insoluble cellular For example, using the method of the present invention, material can be solubilized and the enzyme provided in pET11a-BACE SEQ ID NO:5 and pGE80L-BACE SEQ reduced form by washing in 10 mM Tris HCl buffer (pH 8), 1 ID NO:7 were denatured in the presence of a reducing agent mM EDTA (TE). Inclusion bodies are then extracted with 7.5 at pH 10.5-10.8 as described in Example 4. This solution was Murea, 100 mM BME, and 100 mM AMPSO (3-(1,1-dim diluted with the reducing agent until the absorbance of the ethyl-2-hydroxyethyl)amino-2-hydroxypropanesulfonic solution was 0.4-0.7 at 280 nm. The solution was then diluted 10 about 20-25 fold with cold water and allowed to incubate in a acid, Sigma Cat. No. A 1911) at pH 10.5-11.0. After centrifu cold room for 3-4 days. This method produced an enzyme gation, the protein concentration of the Supernatant can be having an activity, compared to BACE expressed in CHO adjusted by dilution with a solution of the denaturant, e.g. 7.5 cells, of about 57% for pQE80L-BACE SEQID NO:7 and Murea, in a suitable buffer 100 mMAMPSO to read approxi about 47% for pET11a BACE SEQ ID NO:5). Using this mately 0.1 to 4.0, preferably about 0.5 to 3.0 at Aso. Other 15 method, pCRE70-BACE SEQID NO:3 exhibited about 97% buffer solutions can be substituted for AMPSO, such as Tris activity compared to CHO-BACE SEQ ID NO:23), but it HCl or CAPS (3-cyclohexylamino-1-propanesulfonic acid, required 6 weeks of incubation to achieve this activity. Sigma Cat. No. C-2632). In order to shorten the incubation time for pOE70-BACE SEQ ID NO:3, a series of experiments was performed as Refolding of Isolated Recombinant Enzyme described in Example 5. In a first experiment, the absorbance Once the protein has been solubilized, it can be refolded of the solubilized BACE solution was adjusted to 1.5 at 280 into the correct conformation to provide active enzyme. Typi nm before the Solution was split into the three samples having cally, refolding of an expressed recombinant enzyme can be their pH adjusted to 10.0, 10.5 and 11.0. Each of these accomplished by removing the Solubilizing agent and replac samples was then split into ten portions and diluted from 20 to ing it with an aqueous buffer, for example, by dialysis or 25 150 fold. While activity was observed for every dilution, it dilution. Generally, for proteins with disulfide bridges, oxi was observed that the best activity was achieved with a 20 fold dation of the reduced protein occurs prior to or concomitant dilution, while 35 and 50 fold dilutions also provided an with refolding. enzyme having Superior activity. Generally, refolding of recombinant BACE is accom In a second experiment to optimize the refolding procedure plished by permitting the diluted enzyme solution (at about 30 for pGE70-BACE SEQ ID NO:3), protein concentrations pH 10-11) to incubate in a cold room (1° C. to abut 15°C.) for reading 0.5, 1.5 and 3.0 at 280 nm were split into three about three days to several weeks. The time of the incubation samples and diluted 20, 35 and 50 fold. As shown in FIG. 5, depends upon the construct used to express the recombinant it was observed that the best activity was achieved by starting BACE. For example, it has been found that the BACE lacking with an absorbance of 3.0 and diluting 50 fold, while dilutions its prosequence requires from 3 weeks to about 6 weeks of 35 of 20 and 35 fold also provided an enzyme having superior incubation to achieve maximal enzyme activity, while BACE activity. sequences having an intact prosequence require about 2 to 6 Purification of Refolded Enzyme The refolded enzyme can be purified using standard liquid days of incubation. chromatography techniques, such as, for example, cation or As shown in the Examples below, solubilized, recombinant 40 anion exchange chromatography (available, for example, BACE can be diluted in water (about 10-150 fold), preferably from Amersham Pharmacia Biotech), hydrophobic interac to a final concentration of approximately 5 to 50 micrograms tion (available, for example, from Toso Haas), dye interaction BACE per ml of solution, and generally at a pH of about (available, for example from Sigma), ceramic hydroxyapatite 10.5-10.8. This mixture is maintained at temperatures of (available, for example, for Bio-Rad), affinity chromatogra approximately 1° C. to approximately 15° C. for several days 45 phy (for example, using an inhibitor that binds active or weeks and assayed periodically for enzymatic activity. enzyme), or size exclusion chromatography (for example, Activity assays can be performed starting at any time. Gen Sephacryl-S100 or S200 column purification as well as resins erally, activity of the re-folded enzyme will be apparent at from BioRad, Toso Haas, Sigma, and Amersham Pharmacia about 20 to 24 hours after the initial dilution step. Biotech). One or a combination of these purification tech In one aspect of the invention, the solubilized BACE, prior 50 niques can be used according to the invention to provide to dilution, has an absorbance reading of about 0.1 to about purified, recombinant BACE. Anion exchange chromatogra 4.0 at 280 nm. Upon dilution of about 10 to about 150 fold, the phy using, for example, Q-Sepharose, Mono-Q, or Resource concentration of the enzyme is expected to be in the range of Q column purification provides useful separation. about 5-50 micrograms/ml. However, it is expected that other For example, following incubation, the refolded BACE can higher or lower concentrations will produce an active protein. 55 be loaded onto anion exchange column, for example a Q-SE For example, it is expected that the recombinant BACE PHOSE, optionally followed by a column for size exclusion, enzyme will properly refold in concentrations from about 1 for example Sephacryl. As shown in FIGS. 6 and 7, while microgram/ml to about 300 micrograms/ml. Thus, the absor these procedures remove nucleic acids and afford more than a bance of the solubilized BACE solution may be higher or 100 fold concentration of refolded enzyme, there is no sig lower, and the Solution may be diluted to a greater or lesser 60 nificant reduction inforeign protein. Thus the sample may be extent than 10-150 fold, depending upon the starting concen further purified with affinity chromatagraphy. In a preferred tration (as shown by the absorbance or otherwise) of the embodiment, the inhibitor for affinity chromatography is solubilized BACE. The extent of protein activity of the inhibitor I-1 as further described in Example 7. refolded protein can be readily determined using the activity assay described herein. Accordingly, for the purposes herein, 65 Optional HIV-1 Protease Treatment of Recombinant Enzyme dilution of the solubilized BACE refers to the process of The refolded BACE polypeptide can be treated with a diluting a solution of solubilized BACE to provide a concen cleavage enzyme, for example, HIV-1 protease. It has been US 7,732,183 B2 11 12 found that treatment with HIV-1 protease results in cleavage plary reporter proteins include a fluorescing protein (for of BACE at two sites, a major cleavage site and a minor example, green fluorescing proteins, luciferase, and the like) cleavage site. N-terminal amino acid sequencing demon or an enzyme that is used to cleave a substrate to produce a strated that the major cleavage occurs between amino acids colorimetric cleavage product. Also contemplated are tag F’ and V' of BACE, and the minor cleavage occurs between 5 amino acids Y''' and A'. Presently it is understood that the sequences that are commonly used as epitopes for quantita minor cleavage concerns only unfolded BACE and, therefore, tive assays. Preferably, the detectable markers do not interfere Such cleavage removes inactive enzyme. After treatment with with binding of BACE to the substrate, or subsequent cleav HIV-1 protease, the enzyme BACE polypeptide can be sub age of the Substrate. For example, detectable markers can be jected to additional purification steps. As shown in the 10 provided in a suitable size that does not interfere with BACE Examples, treatment with HIV-1 protease can result in a activity. In some embodiments, detectable markers can be 5-fold to 9-fold increase in enzyme activity versus untreated coupled to the Substrate using spacers. enzymes expressed from constructs B1 and B2. According to Table 2 shows a comparison of yields and activities of some embodiments of the invention, treatment with HIV-1 recombinant BACE expressed in CHO cells and E. coli (re protease can produce a homogeneous preparation of BACE 15 with residue V' at its N-terminus SEQID NO:31). ferred to 10 liters of cell culture) for the constructs B1 (SEQ Cleavage with HIV protease can be performed, for ID NO:51, B2 SEQ ID NO:7 and B4 SEQ ID NO:3), example, under the following conditions: pH 4-7, 0.5M-3 M including enzymes Subjected to HIV protease. urea, up to 2 hours incubation with HIV-1 protease, 8.7x10 molar and BACE, 1.705x10 molar, at 37° C. In one TABLE 2 example, HIV-1 protease cleavage can be performed at pH Expression BACE Amount Activity 5.7, 0.5 M Urea, 5% molar concentration HIV-1 protease Source Construct Purified (mg) % (4.08% w/w), BACE, about 2.5 mg/ml, for one hour at 37° C. CHO PcDNA3.1Asp2LATM(His) 19 100 Activity of Refolded BACE SEQ ID NO:23) 25 CHO PcDNA3.1Asp2LATM(His) + 13 120 Activity of the refolded, purified recombinant BACE can HIV Pr(SEQ ID NO:32) be determined by incubating the refolded enzyme with a E. coi pET11a-BACE SEQID NO: 5 55 47 suitable substrate under conditions to allow cleavage of the E. coi ET11a-BACE - HIV Pr 28 248 substrate. The substrate can be labeled with a detectable SEQ ID NO:31) E. coi pOE8OL-BACE SEQID NO: 7 103 57 marker, such as a fluorescent label, to allow detection of 30 E. coi pOE8OL-BACE + HIV Pr 65 222 cleavage events. SEQ ID NO:31) Suitable substrates are peptides that include a BACE cleav E. coli pOE70-BACE SEQID NO:3) 45 97 age site. For example, the synthetic peptides SEISY-EVEFR (from Example 4) E. coi pOE70-BACE SEQID NO:3) WKK SEQ ID NO: 9) and GLTNIKTEEISEISY-EVEFR (from Example 5 -- “optimized WKK SEQID NO: 10 can be cleaved by the recombinant 35 refolding method’): BACE (site marked by “-”). Additional substrates suitable for first elution S4 171 BACE cleavage include SEVNL-DAEFRWKK SEQ ID Second elution 19 125 NO: 11 and GLTNIKTEEISEVNL-DAEFRWKK SEQID NO: 12, containing the APP Swedish Mutation. Table 2 shows a comparison of the yields and activities for The substrate can be labeled with a suitable detectable 40 marker to permit visualization of cleavage. Assays to detect pOE70-BACE(R...S.) SEQID NO:3 to those obtained activity of recombinantly produced BACE can measure reten for pET11a-BACE(A . . . S) (SEQ ID NO:5 and tion or liberation of the detectable marker. Suitable detectable pOE80L-BACE SEQID NO:7). Also, the yield of VE . . . markers include, for example, radioactive, enzymatic, chemi ES’ fragment SEQID NO:31 obtained by HIV protease luminescent, or fluorescent labels. In some embodiments, the 45 treatment of pET11a-BACE SEQ ID NO:5 and pOE80L Substrate can include internally quenched labels that result in BACE SEQID NO:7 is given along with that of the recom increased detection after cleavage of the substrate. The sub binant enzyme from CHO cells (control). The pET11a-BACE strate can be modified to include a paired fluorophore and SEQ ID NO:5 and pCE70-BACE SEQ ID NO:3 con quencher including, but not limited to, 7-amino-4-methyl structs expressed in E. coli and purified to homogeneity by the coumarin and dinitrophenol, respectively, such that cleavage 50 affinity column had 47% and 171% activity, respectively, of the substrate by BACE results in increased fluorescence as relative to the CHO cell derived enzyme. The fragment V" a result of physical separation of the fluorophore and E. . . ES''' BACE SEQID NO:31 obtained by treating the quencher. Other paired fluorophores and quenchers include pET11a-BACE SEQ ID NO:5 construct with the HIV-1 bodipy-tetramethylrhodamine and QSY-5 (Molecular protease was about 5 times more active than the starting Probes, Inc.). 55 construct. N-terminal sequencing and compositional analysis In a variant of this embodiment, biotin or another suitable tag can be placed on one end of the peptide to anchor the confirmed the absence of HIV protease in the sample. peptide to a Substrate assay plate, and a fluorophore can be Additional comparisons of pCRE70-BACE SEQID NO:3) placed at the other end of the peptide. Useful fluorophores from E. Coli, and BACE expressed in CHO cells include those listed herein, as well as Europium labels such as 60 (PcDNA3.1Asp2LATM(His) as described herein, have been W8044 (EG&GWallac, Inc.). One exemplary label is Oregon conducted. Specifically, the two enzymes were compared for green that can be coupled to a cysteine residue. Cleavage of V. k.ef and K. As shown here, the two forms of BACE the substrate by BACE will release the fluorophore or other exhibited similar kinetic parameters. Assays were carried out tag from the plate, allowing detection of an increase in in 10 mM of each sodium acetate and MES, pH 5.5, 50 mM retained fluorescence. 65 NaCl, and substrate concentrations from 1 to 300 uM, at 37° Further examples of detectable markers include a reporter C. for 1 h with enzyme concentrations of 1 nM. The results are protein amino acid sequence coupled to the Substrate. Exem shown in Table 3. US 7,732,183 B2

TABLE 3

Vmax k Kn kcal/Kn Source Substrate Sequence (umol min 'mg (s ec (uM) (secuM) CHO GLTNIKTEEISEISY--EVEFRWKK O. 435 O. 345 4.9 O. Of O4 E. coli SEQ ID NO: 10 O. 4 OO O. 317 5.4 O. O581

CHO SEISY--EVEFRWKK O .294 O. 233 52. O O. OO 45 E. coli SEQ ID NO: 9 O. 345 O. 273 55.5 OOO49

CHO EIDL. --MVLDWHDR O. O.196 O. O155 12.. O O. OO13 E. coli SEQ ID NO: 13 ND ND

Having generally described the invention, the same can be 15 on addition of IPTG. The DNASEQID NO.4 and predicted more readily understood by reference to the following amino acid sequence SEQ ID NO:5 of this construct is examples, which are provided by way of illustration and are shown in FIGS. 2A-2C. not intended as limiting. B2 pCE80L-M-R-G-S-(H)-G-S-I-E-T-D-(T' ... S''') SEQID NO: 7), referred to below as pGE80L-BACE, and in EXAMPLES Table 1 as construct B2. The DNA insert of this construct Example 1 encodes a BACE fragment that is truncated at both the N-ter minal and C-terminal regions. The encoded fragment lacks the transmembrane domain as well as the pre-sequence the Preparation of BACE Constructs B1 and B2 25 BACE protein shown in FIG. 1 SEQ ID NO:1). This con Expression constructs for producing recombinant BACE struct further contains nucleotides encoding a Caspase-8 rec fragments in E. coli were prepared using the vector pGElla ognition site, IETD (bolded) SEQ ID NO:30, enabling from Novagen and vectors pOE80L and pCE70 from Qiagen. cleavage of the expressed BACE polypeptide at the T' posi The nucleotide sequence encoding for BACE was amplified tion, as well as a His purification tag. The construct also by PCR from a full length cDNA template as described in WO 30 includes the T5 lac promoter, permitting induced expression 00/17369, which is incorporated herein by reference in its on addition of IPTG. The DNASEQIDNO:6 and predicted entirety. DNA encoding the desired human BACE polypep amino acid sequence SEQ ID NO:7 of this construct is tide, modified by site-directed mutagenesis to contain pre shown in FIGS. 3A-3C. ferred codons for expression in E. coli were inserted into the B4 pGE70-(M)RGSFVE . . . QTDES'RS(His) 35 SEQ ID NO: 7), referred to below as pCRE70-BACE and in vectors. Expression constructs B1 SEQ ID NO:4, and B2 Table 1 as construct B4. The DNA insert for this construct SEQ ID NO:6 (See Table 1) were developed using frag encodes a BACE fragment that is truncated at both the N-ter ments of this modified codon sequence, having codon minal and C-terminal regions. The encoded fragment lacks changes as compared with the unmodified sequence encoding the transmembrane domain as well as both the pre-sequence BACE, shown in bold type in FIGS. 2A SEQID NO.4 and 40 and pro-sequence of BACE shown in FIG. 1 SEQID NO:1. 3A SEQID NO:6). A codon for methionine is inserted adjacent the first BACE Three specific BACE constructs (B1, B2, and B4) were codon to facilitate expression in prokaryotic cells. A C-ter produced: minal polyhistidine is provided by the vector. The DNASEQ B1. T7 tag (MASMTGGQQMGR)-GSM-BACE ID NO:2 and predicted amino acid sequence SEQID NO:3 (AGVLP . . . TQTDES) (SEQ ID NO. 5), referred to 45 of this construct is shown in FIGS. 4A and 4.B. below as pET11a-BACE, and in Table 1 as construct B1. The Production of pET11a-BACE. To produce the B1 con DNA insert of this construct encodes a BACE fragment that is struct, pET11a-BACE: T7 tag (MASMTGGQQMGR)- truncated at both the N-terminal and C-terminal regions as GSM-BACE (AGVLP ... TQTDES-2) SEQ ID NO:5), the DNA insert was amplified by PCR in a series of steps. compared with the sequence shown in FIG. 1 SEQID NO:1. 50 BACE-encoding polynucleotides 1 to 580 were amplified The insert encodes a protein lacking the transmembrane from a pET11a-BACE construct SEQ ID NO:4) that was domain and a portion of the pre-sequence (leader sequence) truncated at the C-terminus and contained only 4 cysteine domains of BACE, shown in FIG. 1 SEQID NO:1. A codon residues. This template was chosen because it contained for methionine is inserted adjacent the first BACE codon to nucleic acid sequences upstream from pCE 80L-BACE con facilitate expression in prokaryotic cells. The construct also 55 struct SEQ ID NO:6 and also included codon changes for includes the T7 lac promoter, permitting induced expression preferred expression in E. coli. The PCR primers were:

PF35" ggcaggatcc atg got gigt gtt c to coa gct ca 3" (forward), SEQ ID NO: 14 and

PR45 ' td.cc act gtc. cac aat gct c 3' (reverse) . SEQ ID NO: 15 US 7,732,183 B2 15 16 An overlapping segment including the rest of the C-termi- The PCR reaction contained 100 ng of plasmid DNA tem nal amino acids was amplified in a separate PCR, using prim- plate, 100 ng of each primer, 0.25 mM each dNTPs, and 2 CS units of Pfu DNA Polymerase in the buffer provided by the

PR55 ggcaggat.cct a tiga citc atc tdt citg tog aat 3" (reverse) SEQ ID NO: 16 and PF65' g agc att gtg gac agt ggc a 3" (forward). SEQ ID NO: 17 10 The products obtained from these two PCR amplifications manufacturer. Cycling parameters included an initial dena were joined together in a third PCR amplification using the turation at 92° C. for 5 min. followed by 25 cycles of 30 sec external primers PF3 and PR5. This final product was gel denaturation at 92°C., 30 sec. annealing at 60° C. and 2 min. purified, digested with BamHI and ligated into the corre extension at 72°C., plus a final cycle with a 5 min. extension sponding site of vectorpOE 11a. The complete DNASEQID 15 at 72°C. The PCR product was fractionated in a 1% agarose NO:4 and amino acid sequence SEQ ID NO:5 for the gel. The desired band was excised and purified by Geneclean. pET11a-BACE construct is shown in FIGS. 2A-2C. The first The purified fragment was digested with SphII and Bgl II, fifteenamino acids (underlined) correspond to the vector's T7 tag and contain a BamHI cloning site as well as an additional purified in the same manner and ligated to the corresponding methionine. Codon changes as preferred for expression in E. 20 sites of the vector pQE70. coli are shown in bold type. PCR conditions were as follows: one initial cycle of denaturation at 95°C., 30 seconds, 30 Example 2 cycles of 30 seconds denaturation at 95°C., 30 seconds annealing at 60°C., 2 minutes extension at 72°C., followed Transformation of Host Cells, Cell Incubation and Analysis. by one cycle of 5 minutes at 72°C. The reaction components 25 The expression constructs produced BACE polypeptide as :dNT"Fo 1Xcloned Pfu ng E.pol buffer y (StratNENA 1OOLLM N inclusion bodies. The inclusion bodies developed after 2T1 (20 units) of cloned Pful DNA polymerase. approximately two hours of induction, and, due to their large Production of pOE80L-BACE: Primers used to amplify the size, were readily visible by light microscopy. BACE DNA insert for pGE80L-BACE: MRGS (His)-GS- 30 Expression of constructs B1 and B2 and analysis of cell IETD-BACE (TQH ... TDES-2) SEQID NO:7 were: pellets: For constructs B1 SEQID NO.4 and B2 SEQ ID

PF9 5' gctaaggat.cc atc gag acc gac acc caa cat gigt att cqt ctd. c SEQ ID NO: 18 PF1O 5' gctaaggat.cc atc gag acc (forward); SEQ ID NO: 19 and PR11 5' ggcaa.gctt atc atg act cat ctd tot gtg gaa td (reverse) . SEQ ID NO: 2O 40 PCR reagents and conditions were the same as described NO:6), ligated DNA was transformed into E. coli DH5C. for above. The PCR product was gel purified, digested with propagation and DNA isolation, and into E. coli BL21 BamHI/HindIII and ligated to the corresponding sites of vec- CodonPlus Rp (for construct B2) and CodonPlus (DE3) Rip tor pGE80L. The resulting construct is shown in FIGS. (for construct B1) for expression. Transformed cells were 3A-3C SEQID NO:6). A caspase-8 cleavage site and nucle- 45 inoculated into and grown in Luria Broth, pH 7.5 containing otide changes according to E. coli codon preference are - - - - h th leotid fFIG. 3ASEOID NO:6 100 ug/ml ampicillin and 34 g/ml chloromphenicol, at 37° inSnown boldtype. 1n une Thenucleoude N-terminal sequence vector o derived sequence,SEQ includ C. and shaken at 200 rpm (2.5 inch throw). A loop of a ing the polyhistidine handle and BamHI cloning site is under- glycerol Stock of the construct was inoculated into the media. lined. PCR conditions and reagents were the same as those for 50 When the absorbance of the culture at 550 nm reached the production of pET11a-BACE SEQID NO:4). approximately 0.5-0.6, cells were collected by centrifugation, Construction of pCRE70-BACE resuspended in fresh media, and used as inoculum for a sec The nucleotide SEQID NO:2 and predicted amino acid ondary culture at a 1:100 dilution. When cell density reached sequence SEQID NO:3 of the pOE70-BACE construct is Asso-0.5-0.6, cells were harvested by centrifugation at room given in FIGS. 4A and 4.B. The codon for R was changed temperature and then resuspended at the same concentration from aag to cgt, in order to conserve the vector's SphI cloning in fresh LB, again containing amplicillin and chlorompheni site. Primers specific to the desired BACE sequence were col. BACE expression was induced by the addition of IPTG to designed as follows: a final concentration of 1 mM. Expression of the recombinant

AMM 51O 5'ggctgc atg cqt ggc agc titt gtg gag at g g to g (forward), SEQ ID NO. 21 and

AMM 514 5' ggctagat ct tda ct c atc tdt citg togg aa (reverse) . SEQ ID NO. 22 US 7,732,183 B2 17 18 protein was continued for 3 hours after induction (Asso-1.8- 100 ml ofLB plus antibiotics. The culture was amplified at 30 2.0). Cells were collected by centrifugation and stored at -80° or 37°C. to a density of Asso-0.6-0.7, and induced with IPTG C. to a final concentration of 1 mM. Expression was allowed to When expression was conducted under the same condi proceed for 2-3 hours after induction, and the cells were tions but in E. coli BL21 or BL21 (DE3) cells that lack the harvested by centrifugation. The cell pellet was resuspended pACYC-based plasmid encoding rare E. coli codons for argi in TE (10 mM Tris HCl pH 8.0, 1 mM EDTA), cells were nine (aga, agg) and proline (ccc), the inclusion bodies pro disrupted by sonication, and products were analyzed by SDS duced were smaller and did not refract light. Although Suc PAGE followed by COOMMASSIER blue staining. The cessful expression of BACE constructs has been reported in band presumed to contain the BACE protein was blotted to BL21 cells (Tang 2001), increased yield and higher quality 10 PDVF membrane and submitted for N-terminal sequencing. inclusion bodies have been observed when using the Codon Large-scale preparations were performed in multiple 4-liter Plus cells in these studies (data not shown). The CodonPlus flasks, following the same conditions as for the Small-scale cells used herein perform well when induced at a density of experiments, at 37°C. approximately 0.5 (Asso). Example 3 Initially cultures were prepared in 100 ml of media con 15 tained in 500 ml flasks. This process was easily adaptable to a larger flask size and was scaled-up as follows. To shorten the Inclusion Body Harvest overall length of the process, the inoculation rate for the The collected centrifuged cells (cell paste) was resus secondary culture was increased 4-fold to a 1:25 dilution of pended in TE (10 mM Tris HCl pH8.0, 1 mM EDTA) at 1/10 the primary seed culture. Cultures were grown in 1.25 L of of the original culture volume and sonicated. The soluble media contained in 2.8 L. Fernbach flasks and the shaking rate protein fraction was separated from cell debris and insoluble was increased to 225 rpm. proteins by centrifugation at 10,000xg for 15 minutes. Pro Expression of BACE was also evaluated under conditions tein in each of the fractions was analyzed by SDS-PAGE. where the step of replacing fresh culture medium prior to To obtain inclusion bodies, cultured cells expressing con induction of expression was eliminated. Under these condi 25 structs B1 SEQ ID NO:5 and B2 SEQ ID NO:7 were tions, cells were grown to an absorbance of approximately centrifuged to pellet the cells. Cell pellets were weighed from 0.75-0.85 (Asso) prior to induction with IPTG, and cells were 10 liters of cell culture. The wet weight of the cell pellet harvested after approximately 2.5 hours of incubation. Media corresponding to 10 liters of cell culture was 23.5 g. The cell change before induction resulted in cells having uniform pellet was resuspended in 4.0–5.0 ml of 10 mM Tris HCl, 1 morphology at the end of the induction period, but does not 30 mM EDTA, pH 8.1 (TE) buffer per gram of cell pellet. The appear to be required. re-suspended cell pellet was subjected to 16,000 psi in a Cell pellets were resuspended in TE buffer (10 mM Tris French press, or to 12,000 psi in a Rannie apparatus. The HCl, pH 8.0, 1 mM EDTA) at a 1 in 10 dilution of the original resulting solution was centrifuged at 8600 rpm (8840xg) for culture volume and sonicated. The soluble protein fraction 30 minutes in a SS34 rotor. The pellet, including insoluble was separated from the cell debris and insoluble protein frac 35 material, was washed one time in 10 mM Tris HCl buffer, pH tion by centrifugation at 10,000xg for 15 minutes. The frac 8.1, 1 mM EDTA (TE) and centrifuged at 2900 rpm (1000xg) tions were analyzed by SDS PAGE. Total cells or protein for 30 minutes in the SS34 rotor. The wet weight of the fractions (Asoo approximately 0.1) were resuspended in inclusion bodies was 2.67 g. The inclusion bodies were either SDS denaturing buffer, boiled for five minutes, and fraction frozen at -20° C. for use at a later time or immediately ated in a 4-20% gradient gel. A representative gel is shown in 40 extracted as described in the further Examples. FIG. 8. Lanes 3-6 contained protein expressed from the B2 For constructB4SEQIDNO:3), the wet weight of the cell construct SEQ ID NO:6), and lanes 7-10 contain protein pellet corresponding to 10 liters of cell culture was about 23 expressed from B1 SEQID NO:4). Lanes 3 and 7 show total g. The cell pellet was resuspended in 5.0 ml of 10 mM Tris protein obtained from uninduced cells; lanes 4 and 8 show HCl, 1 mM EDTA, pH 8.1 (TE) buffer per gram of cell pellet total protein from induced cells; lanes 5 and 9 show soluble 45 and subjected to 12,000 psi in a Rannie apparatus. The result protein fractions; and lanes 6 and 10 show insoluble protein ing solution was centrifuged at 7400 rpm (8907xg) for one fractions. A prominent band at about 50 kDa, the expected hour in GSA rotor. The pellet was washed one time in TE molecular mass of the encoded BACE fragments, appears in buffer and centrifuged at 2900 rpm (1368xg) for one hour in lanes 6 and 10, in the insoluble protein fractions. the GSA rotor. The inclusion bodies were either frozen at The presence and correct orientation of the insert for the 50 -20°C. for use at a later time or immediately extracted. pET11a construct was confirmed by PCR, using one of the internal primers (PF6 or PR9) and the corresponding flanking Example 4 primer from the pET11a vector. DNA was subjected to com plete sequencing to confirm sequence inserts. Refolding of Recombinant BACE. Expression of construct B4 and analysis of cell pellets: For 55 Protein from the inclusion bodies was solubilized with the transformation and expression of construct B4 SEQ ID 15-20 ml 7.5 Murea, 100 mM AMPSO, and 100 mM BME, NO:2, pGE70 was transformed into the E. coli expression at pH 10.5-10.8. After centrifugation at 11,900 rpm in the strain BL21 (pREP4) and plated on LB agar containing 100 GSA rotor for one hour, the protein concentration of the ug/ml amplicillin and 25ug/mlkanamycin. Selected colonies supernatant was adjusted by dilution with the above buffer to were amplified, and the isolated plasmid DNA was submitted 60 read approximately 4.0 to 7.0 at Aso. The protein was then to DISC for complete sequencing. The BL21 (pREP4) strain diluted with 8 Murea, 100 mMAMPSO, at pH=10.5-10.8 to is the result of the transformation of transformation of the read about 0.4 to 0.7 at Aso. Extraction has also been carried commercially available B121 (Novagen) with the plasmid out substituting AMPSO with CAPS or Tris HCl, with similar pREP4 (Qiagen), which generate high levels of the lac repres results. Sorprotein to ensure tight control at the transcriptional level. 65 Analysis of the sample in 7.5 M urea by SDS-PAGE An overnight culture from a single colony containing the revealed BACE as the major component of the solubilized pOE70-BACE SEQID NO:2) plasmid was used to inoculate inclusion bodies. BACE migrated as a band of Mr 50,000. US 7,732,183 B2 19 20 Refolding was carried out by a 20-25 fold dilution with Example 6 cold water having a temperature of approximately 4°C.-15° C. Upon dilution, the pH of the sample dropped automatically to 9.5-10.2. The sample was then allowed to rest in the cold Expression of BACE in CHO Cells (Control): room for several days. Activity assays were performed daily 5 As a control, a BACE construct (Asp2-2LATM-His), to monitor protein refolding, generally starting about 18-20 referred to herein as CHO-BACE, encoding the amino acid hours after the 20-25 fold dilution with water. Final pH read sequence shown in FIG. 9 SEQID NO: 23, was expressed in ings were in the range of 9.5-10.2. Results from various CHO cells was purified from about 75 liters of conditioned activity assays (described below) indicated that maximal media. The purification process consisted of successive steps activity was usually reached at day 3 or 4 of incubation (data 10 of tangential flow concentration, ammonium sulfate precipi not shown) for enzyme expressed from constructs B1 SEQ tation, Nickel affinity column, and affinity chromatography ID NO:4 and B2 SEQ ID NO:6). For enzyme expressed (I-1 affinity). The purified enzyme contained an from construct B4 SEQID NO:2), maximal activity was not approximately 50:50 mixture of the isoforms starting at reached until about 6 weeks of incubation. T'QHGIRL . . . and ETDEEPEEPG. ..., numbered as in 15 FIG. 1. SEQ ID NO:1. The two isoforms are generated by Example 5 post-translational cleavage by yet unknown proteases. Activ ity of the CHO-BACE SEQID NO:23 served as a control in the activity analysis of the recombinant BACE from con Optimization of Refolding of pCRE70-BACE structs B1 SEQID NO:51, B2 SEQID NO:7 and B4 SEQ In order to improve the refolding protocol for the enzyme ID NO:3 as described below. from construct B4 SEQ ID NO:3, a series of experiments was performed. In the first optimization step, inclusion bodies Example 7 from 3L of cell culture from expression of pCRE70-BACE SEQ ID NO:3 and pET11a-BACE SEQ ID NO:5 (as a control) were extracted with a solution composed of 7.5 M is Purification of Recombinant BACE urea, 100 mM B-mercaptoethanol (BME), and 100 mM To purify the refolded protein, the pH of the refolded pro AMPSO and split in three portions. Each portion was tein was lowered from approximately 10 to 8.5 with dilute adjusted to a different pH; specifically, pH 10.0, 10.5, and HCl and loaded onto a Q-Sepharose column (5.0x2.8 cm). 11.0, respectively. The portions were centrifuged and their BACE was eluted with 0.75 M NaCl in 0.4 Murea, 10 mM absorbances at Aso determined. The three samples were so Tris HCl buffer, pH8.2. Active BACE was collected. The diluted with 7.5 Murea and 100 mM AMPSO to read 1.5 at sample was dialysed against 20 mM Hepes, pH 8.0. After Also nm while their pHs were kept at 10.0, 10.5, and 11.0, dialysis, the pH of the sample was brought to 5.7 using 1 M respectively. Each of the three solutions was then split in ten sodium MES, pH 5.7 (0.1 M final concentration), and centri different portions. Each portion was diluted with water at fuged at 20,000xg for 30 minutes. The pH of the supernatant 4-15° C. to obtain ten different dilutions for each of the three is was then lowered to 5.0 with 1 M sodium acetate, 1 M sodium samples. Specifically, 20-, 30-, 40-, 50- 60-, 70-, 80-, 90 MES, pH 5.0 (0.2 M sodium acetate, 0.28 M sodium MES, 120-, and 150-fold dilutions were performed. The solutions final concentration). The sample was then centrifuged, but no were set-aside in the cold room. Temperature and pH of the pellet was observed. diluted protein solutions were monitored at day 0 and day 7. The Supernatant was applied to a 10 ml affinity column BACE activity assays (described below) with substrate SEI- a (SULFOLINKTM Coupling Gel, Pierce Cat. No. 204011) in SYEVEFRWKK SEQID NO. 9 were carried out at day 7 an Econo column (BioRad) in an amount of 1 mg/ml of the for each of the dilutions. gel, according to the manufacturers instructions cross-linked In the second optimization step for the expression product with the Inhibitor I-1 SEQID NO:28 (described below) and of construct B4 SEQID NO:2, inclusion bodies from 2L of equilibrated at the same pH as the sample. The column was cell culture were extracted with 7.5 M urea, 100 mM BME 4s washed with 6 column volumes of 0.1 M sodium acetate, 0.1 and 100 mMAMPSO, pH 11.0 and centrifuged. Protein con Msodium MES, pH 5.0. BACE was eluted at pH 8.5 in 0.1M centrations reading 0.5, 1.5, and 3.0 at 280 nm were selected. borate buffer. Each solution was split in three portions that were diluted 20 Results showed that this purification method provided a 35-, and 50-fold respectively, with water at 4-15°C. Progress yield, from 10 liters of E. coli cell culture, of 58 mg of highly of refolding was monitored over a period of three weeks by so purified B1 construct SEQ ID NO:5). An equivalent of 1.8 assaying the samples daily. As a control the same experimen liters of E. coli cell culture was affinity purified yielding 16.5 tal procedure was completed for pBT11a-BACE (construct mg of highly purified B2 construct SEQ ID NO:7). By B1 SEQID NO:5). The results are reported in FIG. 5. extrapolating, 10 liters of E. coli would be expected to yield For large scale refolding, inclusion bodies from the expres about 92 mg enzyme. The three batches of E. coli expressing sion of construct B4SEQID NO:2 were obtained from 60 L 55 construct B4SEQID NO:3), (3.3L, 19L, and 40L, making a of cell culture and washed one time in 935 ml of 10 mM Tris total of 62.3L), and refolded using the procedure described HCl buffer, pH 8.1, 1 mM EDTA (TE). The inclusion bodies above requiring six weeks incubation for optimal activity, were extracted with 600 ml 7.5 Murea, 100 mMAMPSO, and produced about 285 mg of pCRE70-BACE(R... S"). SEQ 100 mM BME, pH 10.8. The extract was stirred at room ID NO:3) temperature for 1 hour before centrifugation. After centrifu- 60 This method was used to purify BACE from 33 liters of E. gation, the protein concentration of the Supernatant was coli cell culture from construct B1 SEQ ID NO:5). This adjusted by dilution with 7.5 Murea and 100 mM AMPSO, yielded 147 mg of highly purified BACE. 17 mg of this pH 10.8 to read 1.5 at Aso Refolding was carried out by a 20 preparation was passed over a Sephacryl-S200 column (2.5x fold dilution with cold water (4-15°C.). Upon dilution, the 130 cm) (Amersham Pharmacia Biotech AB) to assess the pH dropped automatically to 10.0-10.3. The sample was then 65 aggregation state of the enzyme. It was observed that BACE allowed to stand in the cold room. Activity assays were per migrated as a monomer, demonstrating that the affinity puri formed regularly to monitor protein refolding. fication step separated monomeric from aggregated forms. A US 7,732,183 B2 21 22 single peak of active BACE, migrating in the monomer posi tion, was observed (data not shown). TABLE 5-continued In the first purification step (above) involving the Q-SEPHAROSETM Fast Flow column, the sample was con Purification of Recombinant BACE Expressed in E. Coli centrated to remove nucleic acids present in abundance at this 5 Total Activity stage. An 8 liter enzyme sample was loaded onto a 50 column Total Protein (Fluorescence pre-equilibrated with 10 mM Tris HCl (pH 8.2), 0.4 Murea Fraction Volume (ml) (mg) peak area) % Yield and NaCl to bring the conductivity to 0.9 mMhos (to match Sephacryl-200 36 14.4 2.3 x 107 29 the ionic strength of the BACE protein solution). A linear Affinity 30 13.8 2.2 x 107 28 gradient of 0-1.0 MNaCl was applied in the same buffer used 10 to equilibrate the column. Fractions of 5.5 ml were collected. Every fifth fraction was assayed for enzymatic activity as Purification of BACE from 60 L E. coli cell culture was described in Example 8. The data are shown here in Table 4. refolded with the optimized method. Prior to purification, the Fractions were also analyzed by SDS-PAGE (5ul samples). pH of the refolded protein was lowered from about 10 to 8.5 15 with HC1. The solution was loaded onto 200 ml Q-Sepharose TABLE 4 in an INdEX 70 mm column (Amersham Biosciences). The column was pre-equilibrated in 0.4 Murea, 10 mM AMPSO, Q-Sepharose Purification of Recombinant BACE pH 8.5. After the refolded protein was loaded onto the col Fluorescence umn, it was washed with 2L of 0.4 Murea, 10 mM Tris HCl, Q-Sepharose peak area pH 8.2. The column was eluted with 600 ml of 0.75 M NaCl in 0.4 Murea 10 mM Tris HCl buffer, pH 8.2. The eluate was Fraction 5 O Fraction 10 O dialyzed overnight versus 10 L 20 mM HEPES, pH 8.0. The Fraction 15 13 sample, 620 ml, was then removed from dialysis and dropped Fraction 20 2O into 69 ml of 1 M NaMES, pH 5.7 (0.1 M NaMES was the Fraction 25 S41 25 final concentration). After centrifugation (20Kxg) the Super Fraction 30 se700 Fraction 35 690 natant was added to 172 ml of 1 MNa-acetate, 1 MNaMES, Fraction 40 699 pH 5.0 (0.2 M sodium acetate, 0.28 MNaMES was the final Fraction 45 222 concentration). While no precipitation was observed at this Fraction 50 222 step, a slight opaqueness was observed while waiting to go Fraction 55 143 30 over the Subsequent affinity column. Consequently, this solu Fraction 60 78 Fraction 65 70 tion was applied rapidly (by gravity) to a 50 ml affinity col Fraction 67 51 umn (SULFOLINK(R) gel cross-linked with inhibitor I-1 Fraction 70 33 SEQ ID NO:28) equilibrated at the same pH. No opaque Fraction 75 21 ness was observed in the resultant flow-through. After recy Fraction 78 17 35 cling the flow through over the affinity column two more times at a slower rate, the column was washed with 300 ml of For affinity purification, affinity column was generated by 10 mM sodium acetate, 10 mM NaMES, pH 5.0. Recombi coupling 1 mg of Inhibitor I-1 (shown below) per ml of nant BACE was eluted with 167 ml of 0.1 M sodium borate SulfoLinkTM Coupling Gel. buffer, pH 8.5. Following the initial elution, the affinity col

OH O H-Ser-Glu-Val-Asn-NH-CH-CH-CH2-C-Val-Ala-Glu-Phe-Arg-Gly-Gly-Cys-OH

CH

CH3-CH

CH3

A Summary of the activity/protein recovery at each purifi umns wash and flow through from the first elution were cation step for construct B1 SEQID NO:5 is shown in Table re-applied to the same affinity column, recycled 3 more times, 5. 55 washed, and eluted as before. The optimized method of refolding produced a total of 438 TABLE 5 mg of highly active and purified pGE70-BACE(R... S'') SEQID NO:3 from the 60 L of E. coli cell culture. Specifi Purification of Recombinant BACE Expressed in E. Coli cally, 322 mg and 116 mg were obtained from the first and Total Activity 60 second affinity column elutions, respectively. FIG. 7 shows Total Protein (Fluorescence the SDS-PAGE of fractions from pCRE70-BACE (R36 . . . Fraction Volume (ml) (mg) peak area) %Yield S432) SEQ ID NO:3 purification steps. Lanes 1 and 3, Standards. Lane 2, insoluble fraction. Lane 4, post Q column. 20-fold dilution 8,000 Sample too 8.0 x 107 1OO Lane 5, post Q column, after the sample was dialyzed. Lane 6, (pH 8.0) step diluted 65 sample of lane 5 was adjusted to pH 5.7. Lane 7, affinity feed. Q-Sepharose 28O 45.3 3.2 x 107 40 Lane 8, affinity flow through. Lane 9, wash. Lane 10, affinity elution US 7,732,183 B2 23 24 Example 8 Example 10

BACE Activity Assays Cleavage of (CHO) Pro-BACE with HIV-1 Protease Peptide substrates SEISY EVEFRWKK SEQID NO: 9 It has been found that HIV-1 protease is capable of cleaving and GLTNIKTEEISEISY EVEFRWKK SEQ ID NO: 10 BACE between residues F-V'. This cleavage site is within were synthesized by solid-phase technology (arrows indicat the protease domain of the BACE enzyme. In this Example, ing cleavage site). The assay mixture containedina Volume of HIV-1 protease was used to produce a homogeneous prepa 200ul, 0.1 M Sodium acetate, pH 5.0, 0.5-20 nM BACE and ration of BACE with V" as an N-terminus. Optimal condi 25 uM substrate SEISY EVEFRWKK SEQ ID NO:9 or tions for cleavage were determined by examining the N-ter GLTNIKTEEISEISY EVEFRWKK SEQ ID NO:10). The 10 minal sequence of BACE after incubation with HIV-1 reaction mixture was incubated for 2 hours at 37° C.; reac protease in various conditions (data not shown). tions were stopped with 100 ul of 4% TFA in HO. A 50 ul Recombinant BACE expressed in CHO cells was obtained portion was injected into an Agilent 1100 Series HPLC as described above for Example 6. HIV cleavage was accom equipped with an Alltech RocketTM column (7 mm i.d.x53 plished in the following manner. 4.65 ml of BACE was added mm length, Cs, 3 um) pre-equilibrated with 88% reagent A 15 to 1.163 ml of 1 M MES, pH 5.7 (no precipitate was (0.1% TFA in water), 12% reagent B (0.1% TFA in acetoni observed). While stirring, 0.55 ml of 6 Murea was added to trile). The flow rate over this column was 3 ml perminute. The the above solution (some precipitate was observed). HIV-1 products (including uncleaved Substrate) were eluted from protease was added in an amount of 0.222 ml. The mixture the column with the following linear gradients: was incubated for 2 hours at 37° C. After incubation, the mixture was dialyzed versus 0.5 M urea, 0.2 M MES (pH 5.7), 4° C. using a Sepcta/Por 6TM O-4.0 minutes 12-30% B Membrane, MWCO: 50,000 (Spectrum Laboratories, part 4.0-6.0 minutes 30-50% B number 132-544) overnight. After overnight incubation, the 6.0-6.5 minutes SO-90% B 25 sample was changed into a solution containing 10 mMMES 6.5-7.0 minutes 90-12% B (pH 5.7), and 50 mM NaCl, 4°C., and allowed to continue 7.0-8.0 minutes 12% B dialyzing for an additional 8 hours. At this point, the Solution was spun to remove the precipitate, and the OD of the super The Agilent 1100 Series HPLC is equipped with a fluores natant was analyzed by absorbance at 280 nm. It was esti cence detector that allows detection of the substrate disap 30 mated, using a conversion factor of 0.685 mg/mg AU. that pearance and EVEFRWKKSEQID NO:29 product forma approximately 20.4 mg of enzyme was present at this stage. tion. Activity was expressed as Fluorescence peak area The prepared BACE sample obtained from the preceding generated by the liberation of a fluorescent (Tryptophan fluo steps was added to 2.325 ml of 1 M sodium acetate, pH 4.5. rescence) product upon cleavage of Substrate. Fluorescence The sample was then applied to a 2 ml affinity column that had detection is monitored at 348 nM upon excitation at 280 nM. been pre-equilibrated with 0.2M sodium acetate, pH 4.5. The 35 affinity column was prepared by coupling Inhibitor I-1 (1 Example 9 mg/ml of resin) to 2 ml Sulfo-LinkTM. The flow through material was recirculated 2 times before washing the column with 20 mM sodium acetate (pH 4.5), 150 mM. NaCl. The Amino Acid Analysis BACE was eluted into 6 ml of 0.1 M sodium borate, pH 8.5. Microwave hydrolyses using CEM Corporation’s MDS 40 Absorbance at 280 nm indicated a total of 13.6 mg of BACE. 2000 microwave oven were conducted in triplicate. Hewlett The affinity column was re-equilibrated, and the process Packard 300 ul microvials containing approximately 2 ug repeated using the flow through. An additional 6 ml contain protein were placed inside a Teflon(R) PFA digestion vessel ing a total of 5.1 mg was recovered, as determined at absor (CEM Corporation) containing 4 ml of 6 N HCl (Pierce bance measurement at 280 nm. Thus, a combined total of 18.7 Constant Boiling) with 0.5% (volume to volume) phenol 45 (Mallinckrodt). The samples were then alternately evacuated mg of BACE was obtained after treatment of the CHO cell and flushed with N, five times. The protein was hydrolyzed derived BACE with HIV-1 protease, and subsequent purifi using a two-stage process. During the first stage 50% of full cation. power (about 650 W) increased the temperature to 100° C. Purified HIV-1 protease treated BACE was assayed for and held at that temperature for 1 min. Immediately follow 50 activity as described above. A control included untreated ing, 75% power increased the temperature to 150° C. and held BACE. Purified HIV-1 protease treated BACE expressed and that temperature for 25 min. After cooling, the samples were purified from CHO cells, demonstrated 10-20% more enzy dried (Savant SpeedVac). Amino acid analyses were per matic activity than untreated BACE. formed on samples derivatized using 6-aminoquinolyl-N-hy Example 11 droxysuccinimidyl carbamate to yield stable ureas that fluo 55 resce at 395 nm (Waters AccCTag Chemistry Package). The samples were analyzed by reverse phase HPLC on a Hewlett Cleavage of Recombinant BACE Expressed in E. coli with Packard 1100 system and quantification was performed using HIV-1 Protease Hewlett Packard's ChemStation enhanced integrator. BACE was expressed from the construct B1 SEQ ID Automated protein and peptide sequencing was performed 60 NO:4, isolated, and refolded as described above for Example on an Applied Biosystems Procise 494 sequencer. Model 1. The refolded protein was passed over a 50 ml 610A version 2.1 software was employed for data acquisition Q-SEPHAROSETM Fast Flow (Amersham Pharmacia Bio and processing. tech) column. The column was pre-equilibrated with 0.4 M Electrospray mass spectrometry data were collected and urea, 10 mM Tris HCl (pH 8.2), with a conductivity that was processed on a Micromass Quattro II quadrupole mass spec 65 adjusted to 0.9 mMhos with sodium chloride. trometer. Data were transformed to a mass scale using the After loading the material, it was washed with five column micromass MaxEnt deconvolution algorithm. volumes of the same buffer used to pre-equilibrate the col US 7,732,183 B2 25 26 umn. The bound protein was then eluted from the column in brated with 20 mMHepes (pH 8.0), and 50 mMNaCl, and the a single step using four column Volumes of 0.4 Murea, 10 protein was allowed to elute in the same buffer. Fractions mM Tris HCl (pH 8.20), 0.75 M sodium chloride. A protein 42-48 of the elution (where the monomeric form would be content of 137 mg was estimated based on absorbance at 280 expected to be present) were pooled and concentrated to nm. This material was then concentrated to 15 ml, and divided approximately 7.5 ml. According to absorbance at 280 nm, into two aliquots for the following experiments. approximately 23.3 mg (4.64x107 moles) of BACE was For the first aliquot, the BACE sample prepared (post present in the sample. Q-Sepharose) was treated directly with HIV-1 protease as The pH of the sample was adjusted to 5.7 with 1 M MES follows. 7.5 ml of the BACE sample was dropped into 1.875 (pH 5.7) by dropping the sample into the concentrated buffer. ml of 1 MMES (pH 5.7), then spun for 15 minutes at 40,000x 10 The final concentration of MES was 0.2 M. The sample was g. To 68.5 mg (1.365x10 moles) of BACE, 2.6 mg (1.3x spun at 40,000xg for 5 minutes, and the supernatant was 107 moles) of HIV-1 protease was added. A significant pre transferred to a new tube. HIV-1 protease (4.64x107 moles, cipitate formed. Notwithstanding, the sample was stirred and 0.933 mg) was added to the sample, then incubated at 37°C. Set in a water bath at 37° C. for 3 hours, 45 minutes. A sample was taken at 1 hour, 10 minutes for SDS-PAGE 15 After incubation, the sample was removed from the 37°C. analysis. At 1 hour, 50 minutes, the sample was removed from bath, and spun to remove any precipitate formed. The Super the water bath and spun for 5 minutes at 40,000xg. The natant was transferred to a new tube at 4°C., and set aside Supernatant was then transferred to a new tube, and placed on while SDS-PAGE analysis was performed. At this point, the ice while samples were analyzed by SDS-PAGE. It appeared SDS-PAGE analysis indicated that the cleavage of BACE was that approximately two-thirds of the material had been approximately two-thirds complete. Therefore, 0.933 mg of cleaved by the HIV-1 protease. Ninety minutes later, 0.65 mg HIV-1 protease was added, and the sample was returned to the HIV-1 protease (33x10 moles) was added, and returned to 37° C. water bath for an additional 1.5 hours. the 37° C. water bath for an additional 1 hour, 40 minutes. At this point, the sample was removed from the 37° C. After this latter addition, the starting BACE material was water bath, and the pH was adjusted to 8.0 with 3M Tris base. almost fully processed. SDS-PAGE demonstrated that a 25 The sample was then spun to remove any further precipita major (and a minor, but significant) cleavage occurred. N-ter tion. The sample was then applied to a 200 cm Sephacryl minal amino acid sequencing confirmed that the major cleav S-100HR (Amersham Pharmacia Biotech) column (Spec age occurred between residues F-V', and minor cleavage trum, 2.5x200 cm) equilibrated with 20 mMHepes pH 8.0, 50 occurs between residues Y-A". Without intending to be mM NaCl, and the protein was allowed to elute in the same bound by a particular theory, it is believed that the latter 30 buffer. This molecular sieving step resolved the active mono cleavage can be desirable to eliminate unfolded BACE. In meric BACE from its aggregated forms. addition, timed studies show near complete cleavage by 3 Fractions containing monomeric BACE were pooled, and hours, 30 minutes. affinity purified with a resin that had been cross-linked with After the final incubation with HIV-1 protease, the material the Inhibitor I-1 SEQID NO:28), as described above. More was then adjusted to pH 8.0 with 3M Tris base, and applied to 35 specifically, fractions were pooled (approximately 35 ml) and a 130 cm Sephacryl S-100HR (Amersham Pharmacia Bio dropped into 8.75 ml of 1 M sodium acetate (pH 4.5), in order tech) column (Spectrum, 2.5x130 cm) equilibrated in 20 mM to adjust the pH into a range that allows BACE to recognize Hepes (pH 8.0), and 50 mMNaCl, and the protein was eluted and bind the inhibitor. The sample was cycled three times in the same buffer. The molecular sieving step was able to over the 10 ml affinity column before washing with 60 ml of separate unprocessed BACE (contained in fractions 55 and 40 20 mM sodium acetate, pH 4.5, 0.15M sodium chloride. The 56) from the bulk of cleaved BACE (fractions 57-60). enzyme was eluted with 30 ml of 0.1 M sodium borate, pH Fractions 57-60 were pooled (approximately 28 ml), and 85. dropped into 7 ml of 1 M sodium acetate, pH 4.50 (0.2M final The sample was dialyzed against 20 mM HEPES, pH 8.0, concentration). The sample was then applied to a 10 ml affin 50 mM NaCl with one change of the buffer. According to ity column, and affinity purified. The resin had been cross 45 absorbance at 280 nm, the final amount of BACE was 7.59 linked with the Inhibitor I-1, as described above. More spe mg. This was analyzed by amino acid analysis (8.01 mg), as cifically, the sample was cycled three times over the 10 ml well as N-terminal sequencing ('VEMVDNL. . . ). Enzy affinity column before washing with 60 ml of 20 mM sodium matic activity of this preparation was also higher than that of acetate (pH 4.5), and 0.15 M sodium chloride. The enzyme the CHO cells derived enzyme, 222% of the CHO control. was eluted with 30 ml of 0.1 M sodium borate, pH 8.5. 50 The sequential processing and purification of the expressed SDS-PAGE shows that the affinity column separated the BACE polypeptides is demonstrated by polyacrylamide gel V...S. SEQID NO:31) fragment from the A'...S. electrophoresis in FIGS. 6 and 10. FIG. 6 shows the process SEQID NO:33 fragment. The latter fragment was found in ing of polypeptides expressed from the construct B1, the flow through. PET11a-BACE SEQ ID NO.4 following two methods of The sample was characterized by amino acid analysis (5.85 55 purification. Lane A2 shows isolated inclusion body protein; mg), N-terminal sequencing ('VEMVDNL . . . ), electro lane A4 shows the refolded protein purified by Q-sepharose; spray mass spectral determination (ESMS) (43.753.6 theo and lane A7 shows the protein product after I-1 affinity chro retical, 43,753.9 observed), and activity assay. The activity matography. Lane B2 shows isolated inclusion body protein; assay showed 248% activity relative to a human BACE con lane B3 shows refolded protein purified through struct expressed in CHO cells. 60 Q-Sepharose; lane B4 through Sephacryl-200; and lane B5 Results indicated that removal of the fragment preceding shows the protein product after I-1 affinity chromatography. V" and of unfolded protein results in an approximate 8- to FIG. 10 shows the processing of polypeptides expressed 9-fold increase inactivity of the recombinant BACE (28% to from the construct B2, including treatments with HIV-pro 248%). tease. Lane A2 shows inclusion bodies; lane A4 shows post For the second aliquot, 7.5 ml of BACE sample (post 65 Q-Sepharose protein; lane A5 shows protein preaffinity chro Q-Sepharose) was applied to a Sephacryl S-200 (Amersham matography, lane A6 shows the affinity column flow through; Pharmacia Biotech) column (Spectrum, 2.5x100 cm) equili and lane A7 shows the protein product after I-1 affinity chro US 7,732,183 B2 27 28 matography. Lane B2 shows inclusion bodies; lane B4 shows changes, adaptations and modifications can be made therein protein post-Q-sepharose; lane B6 shows protein treated with without departing from the spirit of the invention and the HIV-protease; lane B9 shows protein product after I-1 affinity Scope of the appended claims. The scope of the invention chromatography. Lane C2 shows inclusion bodies; lane C4 shows protein post-Q-Sepharose and Sepacryl-200 purifica should, therefore, be determined not with reference to the tion; lane C6 shows post first I-1 affinity chromatography; above description, but instead should be determined with lanes C7 and C9 shows post-dialysis; lane C10 shows after reference to the appended claims along with their full scope of adjustment to pH 5.7; lane C11 shows protein after HIV equivalents. protease treatment; lane C14 shows the protein product after All publications and patent documents cited in this appli second I-1 affinity chromatography. 10 cation are incorporated by reference in their entirety for all While preferred embodiments of the present invention purposes to the extent they are not inconsistent with the have been described, it should be understood that various teachings herein.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 33

<21 Os SEQ ID NO 1 &211s LENGTH: 5O1 212s. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs SEQUENCE: 1

Met Ala Glin Ala Lieu Pro Trp Lieu. Lieu. Lell Trp Met Gly Ala Gly Val 1. 5 15

Leul Pro Ala His Gly Thr Glin His Gly Ile Arg Luell Pro Tell Arg Ser 25 3 O

Gly Lieu. Gly Gly Ala Pro Lieu. Gly Lell Arg Leul Pro Arg Glu Thir Asp 35 4 O 45

Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Wall Glu Met Wall SO 55 60

Asp Asn Lieu. Arg Gly Lys Ser Gly Glin Gly Tyr Wall Glu Met Thir 65 70

Wall Gly Ser Pro Pro Gn. Thir Lieu. Asn Ile Lieu Wall Asp Thir Gly Ser 85 90 95

Ser Asn Phe Ala Wall Gly Ala Ala Pro His Pro Phe Luell His Arg 105 110

Glin Arg Glin Luell Ser Ser Thir Arg Asp Luell Arg Lys Gly Val 115 12O 125

Wall Pro Thr Gin Gly Trp Glu Gly Glu Luell Gly Thir Asp 13 O 135 14 O

Luell Wall Ser Ile Pro His Gly Pro Asn Wall. Thir Wall Arg Ala Asn Ile 145 15 O 155 16 O

Ala Ala Ile Thr Glu Ser Asp Phe Phe Ile Asn Gly Ser Asn Trp 1.65 17 O 17s

Glu Gly Ile Luell Gly Luell Ala Ala Glu Ile Ala Arg Pro Asp Asp 18O 185 190

Ser Luell Glu Pro Phe Phe Asp Ser Lieu Wall Glin Thir His Wall Pro 195 2 OO

ASn Lell Phe Ser Luell Glin Luell Gly Ala Gly Phe Pro Tell ASn Glin 210 215 22 O

Ser Glu Wall Luell Ala Ser Wall Gly Gly Ser Met Ile Ile Gly Gly Ile 225 23 O 235 24 O

Asp His Ser Leu Tyr Thr Gly Ser Luell Trp Thir Pro le Arg Arg 245 25 O 255

Glu Trp Tyr Glu Wall Ile Ile Wall Arg Wall Glu Ile ASn Gly Glin 26 O 265 27 O US 7,732,183 B2 29 30

- Continued Asp Lieu Lys Met Asp Cys Lys Glu Tyr Asn Tyr Asp Llys Ser Ile Val 27s 28O 285 Asp Ser Gly. Thir Thr Asn Lieu. Arg Lieu Pro Llys Llys Val Phe Glu Ala 29 O 295 3 OO Ala Wall Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu Lys Phe Pro Asp 3. OS 310 315 32O Gly Phe Trp Leu Gly Glu Gln Leu Val Cys Trp Glin Ala Gly Thr Thr 3.25 330 335 Pro Trp Asn Ile Phe Pro Val Ile Ser Leu Tyr Lieu Met Gly Glu Val 34 O 345 35. O Thr Asn Glin Ser Phe Arg Ile Thr Ile Leu Pro Glin Glin Tyr Lieu. Arg 355 360 365 Pro Val Glu Asp Wall Ala Thir Ser Glin Asp Asp Cys Tyr Llys Phe Ala 37 O 375 38O Ile Ser Glin Ser Ser Thr Gly Thr Val Met Gly Ala Val Ile Met Glu 385 390 395 4 OO Gly Phe Tyr Val Val Phe Asp Arg Ala Arg Lys Arg Ile Gly Phe Ala 4 OS 41O 415 Val Ser Ala Cys His Val His Asp Glu Phe Arg Thr Ala Ala Val Glu 42O 425 43 O Gly Pro Phe Val Thr Lieu. Asp Met Glu Asp Cys Gly Tyr Asn Ile Pro 435 44 O 445 Gln Thr Asp Glu Ser Thr Lieu Met Thr Ile Ala Tyr Val Met Ala Ala 450 45.5 460 Ile Cys Ala Leu Phe Met Leu Pro Leu. Cys Lieu Met Val Cys Gln Trp 465 470 47s 48O Arg Cys Lieu. Arg Cys Lieu. Arg Glin Gln His Asp Asp Phe Ala Asp Asp 485 490 495 Ile Ser Lieu. Lieu Lys SOO

<210s, SEQ ID NO 2 &211s LENGTH: 1221 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) . . (1221) 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <222s. LOCATION: (168) ... (168) <223> OTHER INFORMATION: N is A, T, G, or C

<4 OOs, SEQUENCE: 2 atg cgt ggc agc titt gtg gag atg gtg gac aac Citg agg ggc aag tog 48 Met Arg Gly Ser Phe Val Glu Met Val Asp Asn Lieu. Arg Gly Lys Ser 1. 5 1O 15 ggg cag ggc tac tac gitg gag atg acc gtg ggc agc ccc ccg cag acg 96 Gly Glin Gly Tyr Tyr Val Glu Met Thr Val Gly Ser Pro Pro Glin Thr 2O 25 3O

Ct c aac at C ctg gtg gat aca ggc agc agt aac titt gca gtg ggit gct 144 Lieu. Asn. Ile Lieu Val Asp Thr Gly Ser Ser Asn. Phe Ala Val Gly Ala 35 4 O 45 gcc ccc cac ccc titc ctd cat cqn tac tac cag agg cag ctg. tcc agc 192 Ala Pro His Pro Phe Lieu. His Arg Tyr Tyr Glin Arg Gln Leu Ser Ser SO 55 6 O aca tac C9g gac Ctc. c99 aag ggc gtg tat gtg CCC tac acc cag ggc 24 O Thr Tyr Arg Asp Lieu. Arg Lys Gly Val Tyr Val Pro Tyr Thr Glin Gly 65 70 7s 8O

US 7,732,183 B2 33 34

- Continued

385 390 395 4 OO

Cat Cac Cat Cac Cat Caic tala 1221 His His His His His His 4 OS

SEQ ID NO 3 LENGTH: TYPE : PRT ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 3

Met Arg Gly Ser Phe Wall Glu Met Wall Asp ASn Lell Arg Gly Lys Ser 1. 5 1O 15

Gly Glin Gly Tyr Tyr Wall Glu Met Thir Wall Gly Ser Pro Pro Glin Thir 25

Lell Asn Ile Luell Wall Asp Thir Gly Ser Ser ASn Phe Ala Wall Gly Ala 35 4 O 45

Ala Pro His Pro Phe Lell His Arg Tyr Glin Arg Glin Luell Ser Ser SO 55 6 O

Thir Arg Asp Lell Arg Gly Wall Wall Pro Thir Glin Gly 65 70

Trp Glu Gly Glu Lell Gly Thir Asp Luell Wall Ser Ile Pro His Gly 85 90 95

Pro Asn Wall Thir Wall Arg Ala Asn Ile Ala Ala Ile Thir Glu Ser Asp 105 11 O

Phe Phe Ile Asn Gly Ser Asn Trp Glu Gly Ile Lell Gly Luell Ala 115 12 O 125

Ala Glu Ile Ala Arg Pro Asp Asp Ser Luell Glu Pro Phe Phe Asp 13 O 135 14 O

Ser Luell Wall Glin Thir His Wall Pro Asn Luell Phe Ser Luell Glin Luell 145 150 155 160

Gly Ala Gly Phe Pro Lell Asn Glin Ser Glu Wall Lell Ala Ser Wall 1.65 17O 17s

Gly Gly Ser Met Ile Ile Gly Gly Ile Asp His Ser Lell Tyr Thir Gly 18O 185 19 O

Ser Luell Trp Thir Pro Ile Arg Arg Glu Trp Tyr Glu Wall Ile 195

Ile Wall Arg Wall Glu Ile Asn Gly Glin Asp Luell Lys Met Asp 21 O 215 22O

Glu Tyr Asn Tyr Asp Lys Ser Ile Wall Asp Ser Gly Thir Thir Asn Luell 225 23 O 235 24 O

Arg Luell Pro Lys Wall Phe Glu Ala Ala Wall Ser Ile Lys Ala 245 250 255

Ala Ser Ser Thir Glu Phe Pro Asp Gly Phe Trp Lell Gly Glu Glin 26 O 265 27 O

Lell Wall Cys Trp Glin Ala Gly Thir Thir Pro Trp Asn Ile Phe Pro Wall 27s 285

Ile Ser Luell Tyr Lell Met Gly Glu Wall Thir ASn Glin Ser Phe Arg Ile 29 O 295 3 OO

Thir Ile Luell Pro Glin Glin Tyr Luell Arg Pro Wall Glu Asp Wall Ala Thir 3. OS 310 315

Ser Glin Asp Asp Cys Phe Ala Ile Ser Glin Ser Ser Thir Gly 3.25 330 335

Thir Wall Met Gly Ala Wall Ile Met Glu Gly Phe Wall Wall Phe Asp 34 O 345 35. O

US 7,732,183 B2 39 40

- Continued

SO 55 6 O

Met Wall Asp Asn Lell Arg Gly Ser Gly Glin Gly Tyr Wall Glu 65 70 7s 8O

Met Thir Wall Gly Ser Pro Pro Glin Thir Luell ASn Ile Lell Wall Asp Thir 85 90 95

Gly Ser Ser Asn Phe Ala Wall Gly Ala Ala Pro His Pro Phe Luell His 105 11 O

Arg Tyr Glin Arg Glin Lell Ser Ser Thir Arg Asp Luell Arg 115 12 O 125

Gly Wall Wall Pro Thir Glin Gly Trp Glu Gly Glu Luell Gly 13 O 135 14 O

Thir Asp Luell Wall Ser Ile Pro His Gly Pro ASn Wall Thir Wall Arg Ala 145 150 155 160

Asn Ile Ala Ala Ile Thir Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser 1.65 17s

Asn Trp Glu Gly Ile Lell Gly Luell Ala Ala Glu Ile Ala Arg Pro 18O 185 19 O

Asp Asp Ser Luell Glu Pro Phe Phe Asp Ser Luell Wall Lys Glin Thir His 195

Wall Pro Asn Luell Phe Ser Lell Glin Luell Gly Ala Gly Phe Pro Luell 21 O 215

Asn Glin Ser Glu Wall Lell Ala Ser Wall Gly Gly Ser Met Ile Ile Gly 225 23 O 235 24 O

Gly Ile Asp His Ser Lell Thir Gly Ser Luell Trp Thir Pro Ile 245 250 255

Arg Arg Glu Trp Tyr Glu Wall Ile Ile Wall Arg Wall Glu Ile Asn 26 O 265 27 O

Gly Glin Asp Luell Met Asp Cys Glu Tyr Asn Tyr Asp Ser 28O 285

Ile Wall Asp Ser Gly Thir Thir Asn Luell Arg Luell Pro Wall Phe 29 O 295 3 OO

Glu Ala Ala Wall Lys Ser Ile Ala Ala Ser Ser Thir Glu Phe 3. OS 310 315

Pro Asp Gly Phe Trp Lell Gly Glu Glin Luell Wall Trp Glin Ala Gly 3.25 330 335

Thir Thir Pro Trp Asn Ile Phe Pro Wall Ile Ser Lell Tyr Luell Met Gly 34 O 345 35. O

Glu Wall Thir Asn Glin Ser Phe Arg Ile Thir Ile Lell Pro Glin Glin Tyr 355 360 365

Lell Arg Pro Wall Glu Asp Wall Ala Thir Ser Glin Asp Asp 37 O 375

Phe Ala Ile Ser Glin Ser Ser Thir Gly Thir Wall Met Gly Ala Wall Ile 385 390 395 4 OO

Met Glu Gly Phe Tyr Wall Wall Phe Asp Arg Ala Arg Arg Ile Gly 4 OS 415

Phe Ala Wall Ser Ala His Wall His Asp Glu Phe Arg Thir Ala Ala 425 43 O

Wall Glu Gly Pro Phe Wall Thir Luell Asp Met Glu Asp Cys Gly Asn 435 44 O 445

Ile Pro Glin Thir Asp Glu Ser 450 45.5

<210s, SEQ ID NO 6

US 7,732,183 B2 43 44

- Continued

27s 28O 285 aac citt cqt ttg ccc aag aaa gtg titt gaa got gca gtc. aaa to c atc 912 Asn Lieu. Arg Lieu Pro Llys Llys Val Phe Glu Ala Ala Wall Lys Ser Ile 29 O 295 3 OO aag gca gcc toc ticc acg gag aag tt C cct gat ggt titc tig ct a gga 96.O Lys Ala Ala Ser Ser Thr Glu Lys Phe Pro Asp Gly Phe Trp Lieu. Gly 3. OS 310 315 32O gag cag Ctg gtg to t caa goa ggc acc acc cct t aac att tt C OO8 Glu Glin Leu Val Cys Trp Glin Ala Gly Thr Thr Pro Trp Asn Ile Phe 3.25 330 335 cca gtc atc. tca citc tac cta atg ggit gag gtt acc aac cag to c ttic O56 Pro Val Ile Ser Lieu. Tyr Lieu Met Gly Glu Val Thr Asn Glin Ser Phe 34 O 345 35. O cgc at C acc at C Ctt CC9 cag caa tac ctg cgg cca gtg gala gat gtg 104 Arg Ile Thir Ile Lieu Pro Glin Glin Tyr Lieu. Arg Pro Val Glu Asp Wall 355 360 365 gcc acg to c caa gac gac tdt tac aag titt goc atc. tca cag to a to c 152 Ala Thir Ser Glin Asp Asp Cys Tyr Llys Phe Ala Ile Ser Glin Ser Ser 37 O 375 38O acg ggc act gtt atg gga gct gtt at C atg gag ggc titc tac gtt gt C 2OO Thr Gly Thr Val Met Gly Ala Val Ile Met Glu Gly Phe Tyr Val Val 385 390 395 4 OO titt gat cqg gCC cca aaa cqa att ggc titt gct gtc. agc gct tcc cat 248 Phe Asp Arg Ala Arg Lys Arg Ile Gly Phe Ala Val Ser Ala Cys His 4 OS 41O 415 gtg cac gat gag titc agg acg gCa gC9 gtg gaa ggc cct titt gtC acc 296 Val His Asp Glu Phe Arg Thr Ala Ala Val Glu Gly Pro Phe Val Thr 42O 425 43 O ttg gac atg gala gac tt ggc tac aac att coa cag aca gat gag to a 344 Lieu. Asp Met Glu Asp Cys Gly Tyr Asn. Ile Pro Glin Thr Asp Glu Ser 435 44 O 445 tga 347

<210s, SEQ ID NO 7 &211s LENGTH: 448 212. TYPE: PRT <213> ORGANISM: Homo sapiens

<4 OO > SEQUENCE: 7 Met Arg Gly Ser His His His His His His Gly Ser Ile Glu. Thir Asp 1. 5 1O 15 Thr Glin His Gly Ile Arg Lieu Pro Lieu. Arg Ser Gly Lieu. Gly Gly Ala 2O 25 3O Pro Lieu. Gly Lieu. Arg Lieu Pro Arg Glu Thir Asp Glu Glu Pro Glu Glu 35 4 O 45 Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val Asp Asn Lieu. Arg Gly SO 55 6 O Lys Ser Gly Glin Gly Tyr Tyr Val Glu Met Thr Val Gly Ser Pro Pro 65 70 7s 8O Glin Thr Lieu. Asn. Ile Lieu Val Asp Thr Gly Ser Ser Asn. Phe Ala Val 85 90 95 Gly Ala Ala Pro His Pro Phe Lieu. His Arg Tyr Tyr Glin Arg Glin Lieu. 1OO 105 11 O Ser Ser Thr Tyr Arg Asp Lieu. Arg Lys Gly Val Tyr Val Pro Tyr Thr 115 12 O 125 Glin Gly Lys Trp Glu Gly Glu Lieu. Gly. Thir Asp Lieu Val Ser Ile Pro 13 O 135 14 O US 7,732,183 B2 45 46

- Continued His Gly Pro Asn Val Thr Val Arg Ala Asn. Ile Ala Ala Ile Thr Glu 145 150 155 160 Ser Asp Llys Phe Phe Ile Asn Gly Ser Asn Trp Glu Gly Ile Lieu. Gly 1.65 17O 17s Lieu Ala Tyr Ala Glu Ile Ala Arg Pro Asp Asp Ser Lieu. Glu Pro Phe 18O 185 19 O Phe Asp Ser Leu Val Lys Glin Thr His Val Pro Asn Lieu Phe Ser Lieu. 195 2OO 2O5 Glin Lieu. Cys Gly Ala Gly Phe Pro Lieu. Asn. Glin Ser Glu Val Lieu Ala 21 O 215 22O Ser Val Gly Gly Ser Met Ile Ile Gly Gly Ile Asp His Ser Leu Tyr 225 23 O 235 24 O Thr Gly Ser Leu Trp Tyr Thr Pro Ile Arg Arg Glu Trp Tyr Tyr Glu 245 250 255 Val Ile Ile Val Arg Val Glu Ile Asin Gly Glin Asp Lieu Lys Met Asp 26 O 265 27 O Cys Lys Glu Tyr Asn Tyr Asp Llys Ser Ile Val Asp Ser Gly. Thir Thr 27s 28O 285 Asn Lieu. Arg Lieu Pro Llys Llys Val Phe Glu Ala Ala Wall Lys Ser Ile 29 O 295 3 OO Lys Ala Ala Ser Ser Thr Glu Lys Phe Pro Asp Gly Phe Trp Lieu. Gly 3. OS 310 315 32O Glu Glin Leu Val Cys Trp Glin Ala Gly Thr Thr Pro Trp Asn Ile Phe 3.25 330 335 Pro Val Ile Ser Lieu. Tyr Lieu Met Gly Glu Val Thr Asn Glin Ser Phe 34 O 345 35. O Arg Ile Thir Ile Lieu Pro Glin Glin Tyr Lieu. Arg Pro Val Glu Asp Wall 355 360 365 Ala Thir Ser Glin Asp Asp Cys Tyr Llys Phe Ala Ile Ser Glin Ser Ser 37 O 375 38O Thr Gly Thr Val Met Gly Ala Val Ile Met Glu Gly Phe Tyr Val Val 385 390 395 4 OO Phe Asp Arg Ala Arg Lys Arg Ile Gly Phe Ala Val Ser Ala Cys His 4 OS 41O 415 Val His Asp Glu Phe Arg Thr Ala Ala Val Glu Gly Pro Phe Val Thr 42O 425 43 O Lieu. Asp Met Glu Asp Cys Gly Tyr Asn. Ile Pro Glin Thr Asp Glu Ser 435 44 O 445

<210s, SEQ ID NO 8 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: T7- Tag <4 OOs, SEQUENCE: 8 Met Ala Ser Met Thr Gly Gly Glin Gln Met Gly Arg 1. 5 1O

<210s, SEQ ID NO 9 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: US 7,732,183 B2 47 48

- Continued <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Synthetic Peptide Substrate for BACE <4 OOs, SEQUENCE: 9 Ser Glu Ile Ser Tyr Glu Val Glu Phe Arg Trp Llys Llys 1. 5 1O

<210s, SEQ ID NO 10 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Synthetic Peptide Substrate for BACE <4 OOs, SEQUENCE: 10 Gly Lieu. Thir Asn Ile Llys Thr Glu Glu Ile Ser Glu Ile Ser Tyr Glu 1. 5 1O 15 Val Glu Phe Arg Trp Llys Llys 2O

<210s, SEQ ID NO 11 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Synthetic Substrate for BACE <4 OOs, SEQUENCE: 11 Ser Glu Val Asn Lieu. Asp Ala Glu Phe Arg Trp Llys Llys 1. 5 1O

<210s, SEQ ID NO 12 &211s LENGTH: 23 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Synthetic Peptide Substrate for BACE <4 OOs, SEQUENCE: 12 Gly Lieu. Thir Asn. Ile Llys Thr Glu Glu Ile Ser Glu Val Asn Lieu. Asp 1. 5 1O 15 Ala Glu Phe Arg Trp Llys Llys 2O

<210s, SEQ ID NO 13 &211s LENGTH: 12 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Synthetic Peptide Substrate for BACE <4 OOs, SEQUENCE: 13 Glu Ile Asp Lieu Met Val Lieu. Asp Trp His Asp Arg 1. 5 1O US 7,732,183 B2 49 50

- Continued

<210s, SEQ ID NO 14 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Primer: BACE N-terminal forward primer for recombinant pET11a-BACE

<4 OOs, SEQUENCE: 14 ggcaggat.cc atggctggtg ttctgc.ca.gc tica 33

<210s, SEQ ID NO 15 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Primer: BACE N-terminal reverse primer for recombinant pET11a-BACE

<4 OOs, SEQUENCE: 15 tgcc actgtc. cacaatgctic 2O

<210s, SEQ ID NO 16 &211s LENGTH: 33 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Primer: BACE C-terminal reverse primer for recombinant pET11a-BACE

<4 OOs, SEQUENCE: 16 ggcaggat.cc tatgactic at Ctgtctgtgg aat 33

<210s, SEQ ID NO 17 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Primer: BACE C-terminal forward primer for recombinant pET11a-BACE

<4 OOs, SEQUENCE: 17 gaggattgttg gacagtggca 2O

<210s, SEQ ID NO 18 &211s LENGTH: 45 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Primer: BACE primer for recombinant poE8OL-BACE

<4 OOs, SEQUENCE: 18 gctaaggat.c catcgagacic gacacccaac atgg tatt cq t ctgc 45 US 7,732,183 B2 51 52

- Continued

<210s, SEQ ID NO 19 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Primer: BACE forward primer for recombinant pQE8 OL-BACE <4 OOs, SEQUENCE: 19 gctaaggat.c catcgagacic 2O

<210s, SEQ ID NO 2 O &211s LENGTH: 35 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Primer: BACE reverse primer for recombinant pQE8 OL-BACE <4 OOs, SEQUENCE: 2O ggcaa.gctta t catgactica totgtctgtg gaatg 35

<210s, SEQ ID NO 21 &211s LENGTH: 34 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Primer: BACE forward primer for recombinant pOE 70-BACE

<4 OOs, SEQUENCE: 21 ggctgcatgc gtggcagott ttggagatg gtgg 34

<210s, SEQ ID NO 22 &211s LENGTH: 30 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221 > NAMEAKEY: misc feature <223> OTHER INFORMATION: Primer: BACE reverse primer for recombinant pQE70-BACE

<4 OOs, SEQUENCE: 22 ggctagat.ct tact Catct gtctgtggaa 3 O

<210s, SEQ ID NO 23 &211s LENGTH: 459 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <223> OTHER INFORMATION: Human soluble BACE with 6-His tag <4 OOs, SEQUENCE: 23 Met Ala Glin Ala Lieu Pro Trp Lieu. Lieu. Lieu. Trp Met Gly Ala Gly Val 1. 5 1O 15 US 7,732,183 B2 53 54

- Continued Lell Pro Ala His Gly Thir Glin His Gly Ile Arg Lieu Pro Lieu. Arg Ser 25

Gly Luell Gly Gly Ala Pro Lell Gly Luell Arg Luell Pro Arg Glu Thir Asp 35 4 O 45

Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Wall Glu Met Wall SO 55 6 O

Asp Asn Luell Arg Gly Lys Ser Gly Glin Gly Tyr Wall Glu Met Thir 65 70

Wall Gly Ser Pro Pro Glin Thir Luell Asn Ile Luell Wall Asp Thir Gly Ser 85 90 95

Ser Asn Phe Ala Wall Gly Ala Ala Pro His Pro Phe Lell His Arg Tyr 105 11 O

Glin Arg Glin Lell Ser Ser Thir Asp Lell Arg Gly Wall 115 12 O 125

Wall Pro Thir Glin Gly Lys Trp Glu Gly Glu Lell Gly Thir Asp 13 O 135 14 O

Lell Wall Ser Ile Pro His Gly Pro Asn Wall Thir Wall Arg Ala Asn Ile 145 150 155 160

Ala Ala Ile Thir Glu Ser Asp Phe Phe Ile Asn Gly Ser Asn Trp 1.65 17O 17s

Glu Gly Ile Luell Gly Lell Ala Ala Glu Ile Ala Arg Pro Asp Asp 18O 185 19 O

Ser Luell Glu Pro Phe Phe Asp Ser Luell Wall Glin Thir His Wall Pro 195 2OO

Asn Luell Phe Ser Lell Glin Lell Gly Ala Gly Phe Pro Luell Asn Glin 21 O 215 22O

Ser Glu Wall Luell Ala Ser Wall Gly Gly Ser Met Ile Ile Gly Gly Ile 225 23 O 235 24 O

Asp His Ser Luell Tyr Thir Gly Ser Luell Trp Thir Pro Ile Arg Arg 245 250 255

Glu Trp Tyr Glu Wall Ile Ile Wall Arg Wall Glu Ile Asn Gly Glin 26 O 265 27 O

Asp Luell Lys Met Asp Glu Tyr Asn Tyr Asp Lys Ser Ile Wall 27s 28O 285

Asp Ser Gly Thir Thir Asn Lell Arg Luell Pro Lys Wall Phe Glu Ala 29 O 295 3 OO

Ala Wall Ser Ile Lys Ala Ala Ser Ser Thir Glu Phe Pro Asp 3. OS 310 315

Gly Phe Trp Luell Gly Glu Glin Luell Wall Cys Trp Glin Ala Gly Thir Thir 3.25 330 335

Pro Trp Asn Ile Phe Pro Wall Ile Ser Luell Tyr Lell Met Gly Glu Wall 34 O 345 35. O

Thir Asn Glin Ser Phe Arg Ile Thir Ile Luell Pro Glin Glin Tyr Luell Arg 355 360 365

Pro Wall Glu Asp Wall Ala Thir Ser Glin Asp Asp Cys Phe Ala 37 O 375

Ile Ser Glin Ser Ser Thir Gly Thir Wall Met Gly Ala Wall Ile Met Glu 385 390 395 4 OO

Gly Phe Wall Wall Phe Asp Arg Ala Arg Arg Ile Gly Phe Ala 4 OS 415

Wall Ser Ala Cys His Wall His Asp Glu Phe Thir Ala Ala Wall Glu 425 43 O

Gly Pro Phe Wall Thir Lell Asp Met Glu Asp Gly Asn Ile Pro

US 7,732,183 B2 57

- Continued gag tac aac tat gac aag agc att gtg gac agt ggc acc acc aac Ctt 816 Glu Tyr Asn Tyr Asp Llys Ser Ile Val Asp Ser Gly. Thir Thr Asn Lieu. 26 O 265 27 O cgt ttg ccc aag aaa gtg titt gala gct gca gtc. aaa t cc at C aag gca 864 Arg Lieu Pro Llys Llys Val Phe Glu Ala Ala Val Llys Ser Ile Lys Ala 27s 28O 285 gcc to C to c acg gag aag titc cct gat ggit ttctgg cta gga gag cag 912 Ala Ser Ser Thr Glu Lys Phe Pro Asp Gly Phe Trp Lieu. Gly Glu Gln 29 O 295 3 OO

Ctg gtg to tdg caa gCaggc acc acc cct tdgaac att tt C cca gt C 96.O Lieu Val Cys Trp Glin Ala Gly Thr Thr Pro Trp Asn Ile Phe Pro Val 3. OS 310 315 32O atc. tca ct c tac cta atg gigt gag gtt acc aac cag toc ttic cqc atc OO8 Ile Ser Leu Tyr Lieu Met Gly Glu Val Thr Asn Glin Ser Phe Arg Ile 3.25 330 335 acc at C Ctt cog cag caa tac Ctg cgg cca gtg gaa gat gtg gCC acg O56 Thir Ile Leu Pro Glin Glin Tyr Lieu. Arg Pro Val Glu Asp Val Ala Thr 34 O 345 35. O tcc caa gac gac tdt tac aag titt gcc atc. tca cag to a to c acg ggc 104 Ser Glin Asp Asp Cys Tyr Lys Phe Ala Ile Ser Glin Ser Ser Thr Gly 355 360 365 act gtt atg gga gct gtt atc atg gag ggc titc tac gtt gt C titt gat 152 Thr Val Met Gly Ala Val Ile Met Glu Gly Phe Tyr Val Val Phe Asp 37 O 375 38O cgg gcc ca aaa ca att ggc titt gct gtC agc gct tc cat gtg cac 2OO Arg Ala Arg Lys Arg Ile Gly Phe Ala Val Ser Ala Cys His Val His 385 390 395 4 OO gat gag tt C agg acg gca gcg gtg gala ggc cct titt gtc. acc ttg gac 248 Asp Glu Phe Arg Thr Ala Ala Val Glu Gly Pro Phe Val Thir Lieu. Asp 4 OS 41O 415 atg gala gac tdt ggc tac aac att coa cag aca gat gag to a tag 293 Met Glu Asp Cys Gly Tyr Asn Ile Pro Glin Thr Asp Glu Ser 42O 425 43 O

<210s, SEQ ID NO 25 &211s LENGTH: 430 212. TYPE: PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 25 Met Ala Ser Met Thr Gly Gly Glin Gln Met Gly Arg Gly Ser Met Ala 1. 5 1O 15 Gly Val Lieu Pro Ala His Gly Thr Gln His Gly Ile Arg Lieu Pro Lieu. 2O 25 3O Arg Ser Gly Lieu. Gly Gly Ala Pro Lieu. Gly Lieu. Arg Lieu Pro Arg Glu 35 4 O 45 Thr Asp Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Val Glu SO 55 6 O Met Val Asp Asn Lieu. Arg Gly Lys Ser Gly Glin Gly Tyr Tyr Val Glu 65 70 7s 8O Met Thr Val Gly Ser Pro Pro Gln Thr Lieu. Asn Ile Leu Val Asp Thr 85 90 95 Gly Ser Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Lieu. His 1OO 105 11 O Arg Tyr Tyr Glin Arg Glin Lieu. Ser Ser Thr Tyr Arg Asp Lieu. Arg Llys 115 12 O 125 Gly Val Tyr Val Pro Tyr Thr Glin Gly Lys Trp Glu Gly Glu Lieu. Gly 13 O 135 14 O US 7,732,183 B2 59 60

- Continued

Thir Asp Lieu Val Ser Ile Pro His Gly Pro ASn Wall Thir Wall Arg Ala 145 150 155 160

Asn Ile Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser 1.65 17s

Asn Trp Glu Gly Ile Lieu. Gly Lieu. Ala Ala Glu Ile Ala Arg Luell 18O 185 19 O

Gly Ala Gly Phe Pro Lieu. Asn Glin Ser Glu Wall Lell Ala Ser Wall 195 2OO

Gly Gly Ser Met Ile Ile Gly Gly Ile Asp His Ser Lell Tyr Thir Gly 21 O 215 22O

Ser Leu Trp Tyr Thr Pro Ile Arg Arg Glu Trp Glu Wall Ile 225 23 O 235 24 O

Ile Val Arg Val Glu Ile Asn Gly Glin Asp Luell Met Asp Cys 245 250 255

Glu Tyr Asn Tyr Asp Llys Ser Ile Wall Asp Ser Thir Thir Asn Luell 26 O 265 27 O

Arg Lieu Pro Llys Llys Val Phe Glu Ala Ala Wall Ser Ile Ala 27s 28O 285

Ala Ser Ser Thr Glu Lys Phe Pro Asp Gly Phe Trp Lell Gly Glu Glin 29 O 295 3 OO

Lell Val Cys Trp Glin Ala Gly Thr Thir Pro Trp Asn Ile Phe Pro Wall 3. OS 310 315

Ile Ser Leu Tyr Lieu Met Gly Glu Wall Thir ASn Glin Ser Phe Arg Ile 3.25 330 335

Thir Ile Leu Pro Glin Glin Tyr Lieu. Arg Pro Wall Glu Asp Wall Ala Thir 34 O 345 35. O

Ser Glin Asp Asp Cys Tyr Llys Phe Ala Ile Ser Glin Ser Ser Thir Gly 355 360 365

Thir Val Met Gly Ala Val Ile Met Glu Gly Phe Tyr Wall Wall Phe Asp 37 O 375

Arg Ala Arg Lys Arg Ile Gly Phe Ala Wall Ser Ala His Wall His 385 390 395 4 OO

Asp Glu Phe Arg Thr Ala Ala Val Glu Gly Pro Phe Wall Thir Luell Asp 4 OS 415

Met Glu Asp Cys Gly Tyr Asn. Ile Pro Glin Thir Asp Glu Ser 42O 425 43 O

<210s, SEQ ID NO 26 &211s LENGTH: 1236 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: CDS <222s. LOCATION: (1) . . (1236)

<4 OOs, SEQUENCE: 26 atg gct agc atg act ggit gga cag Cala atg ggt cgc gga to c atg gct 48 Met Ala Ser Met Thr Gly Gly Glin Glin Met Gly Arg Gly Ser Met Ala 1. 1O 15 ggit gtt Ctg cca gct cac ggit acc Cala Cat ggt att cgt Ctg CC a Ctg 96 Gly Val Lieu Pro Ala His Gly Thr Glin His Gly Ile Arg Luell Pro Luell 2O 25 3O cgt agc ggit ctg. g.gt ggit gct coa Ctg ggt Ctg cgt Ctg cc c cgg gag 144 Arg Ser Gly Lieu. Gly Gly Ala Pro Luell Gly Luell Arg Lell Pro Arg Glu 35 4 O 45 a CC gac gala gag ccc gag gag ccc ggc cgg agg ggc agc titt gag 192

US 7,732,183 B2 63 64

- Continued a.a.a. cga att ggc titt gct gtc agc gct tgc Cat gtg CaC gat gag ttic 1152 Arg Ile Gly Phe Ala Wall Ser Ala Cys His Wall His Asp Glu Phe 37 O 375 38O agg acg gca gcg gtg gaa ggc cott titt gt C acc gac atg gala gac 12 OO Arg Thir Ala Ala Wall Glu Gly Pro Phe Wall Thir Lell Asp Met Glu Asp 385 390 395 4 OO tgt ggc tac aac att C Ca Cag aca gat gag toa tag 1236 Cys Gly Asn Ile Pro Glin Thir Asp Glu Ser 4 OS 41O

SEO ID NO 27 LENGTH: 411 TYPE : PRT ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 27

Met Ala Ser Met Thir Gly Gly Glin Glin Met Gly Arg Gly Ser Met Ala 1. 5 15

Gly Wall Luell Pro Ala His Gly Thir Glin His Gly Ile Arg Luell Pro Luell 25 3O

Arg Ser Gly Luell Gly Gly Ala Pro Luell Gly Luell Arg Lell Pro Arg Glu 35 4 O 45

Thir Asp Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Wall Glu SO 55 6 O

Met Wall Asp Asn Lell Arg Gly Ser Gly Glin Gly Wall Glu 65 70

Met Thir Wall Gly Ser Pro Pro Glin Thir Luell ASn Ile Lell Wall Asp Thir 85 90 95

Gly Ser Ser Asn Phe Ala Wall Gly Ala Ala Pro His Pro Phe Luell His 105 11 O

Arg Tyr Glin Arg Glin Lell Ser Ser Thir Arg Asp Luell Arg 115 12 O 125

Gly Wall Wall Pro Thir Glin Gly Trp Glu Gly Glu Luell Gly 13 O 135 14 O

Thir Asp Luell Pro Asp Asp Ser Luell Glu Pro Phe Phe Asp Ser Luell Wall 145 150 155 160

Glin Thir His Wall Pro Asn Luell Phe Ser Luell Glin Lell Gly Ala 1.65 17O 17s

Gly Phe Pro Luell Asn Glin Ser Glu Wall Luell Ala Ser Wall Gly Gly Ser 18O 185 19 O

Met Ile Ile Gly Gly Ile Asp His Ser Luell Thir Gly Ser Luell Trp 195

Thir Pro Ile Arg Arg Glu Trp Glu Wall Ile Ile Wall Arg 21 O 215 22O

Wall Glu Ile Asn Gly Glin Asp Luell Met Asp Glu Asn 225 23 O 235 24 O

Asp Ser Ile Wall Asp Ser Gly Thir Thir Asn Lell Arg Luell Pro 245 250 255

Wall Phe Glu Ala Ala Wall Lys Ser Ile Ala Ala Ser Ser 26 O 265 27 O

Thir Glu Lys Phe Pro Asp Gly Phe Trp Luell Gly Glu Glin Luell Wall Cys 27s 28O 285

Trp Glin Ala Gly Thir Thir Pro Trp Asn Ile Phe Pro Wall Ile Ser Luell 29 O 295 3 OO

Tyr Luell Met Gly Glu Wall Thir Asn Glin Ser Phe Arg Ile Thir Ile Luell 3. OS 310 315 32O US 7,732,183 B2 65

- Continued

Pro Glin Glin Tyr Lieu. Arg Pro Val Glu Asp Wall Ala Thir Ser Glin Asp 3.25 330 335 Asp Cys Tyr Llys Phe Ala Ile Ser Glin Ser Ser Thr Gly Thr Val Met 34 O 345 35. O Gly Ala Val Ile Met Glu Gly Phe Tyr Val Val Phe Asp Arg Ala Arg 355 360 365 Lys Arg Ile Gly Phe Ala Val Ser Ala Cys His Val His Asp Glu Phe 37 O 375 38O Arg Thr Ala Ala Val Glu Gly Pro Phe Val Thir Lieu. Asp Met Glu Asp 385 390 395 4 OO Cys Gly Tyr Asn Ile Pro Gln Thr Asp Glu Ser 4 OS 41O

<210s, SEQ ID NO 28 &211s LENGTH: 13 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <222s. LOCATION: (5) . . (5) <223> OTHER INFORMATION: Xaa is 4-amino-3-hydroxy-6-methylheptanoic acid

<4 OOs, SEQUENCE: 28 Ser Glu Val Asn. Xaa Val Ala Glu Phe Arg Gly Gly Cys 1. 5 1O

<210s, SEQ ID NO 29 &211s LENGTH: 8 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <223> OTHER INFORMATION: Cleavage Product from Peptide Substrate <4 OOs, SEQUENCE: 29 Glu Val Glu Phe Arg Trp Llys Llys 1. 5

<210s, SEQ ID NO 3 O &211s LENGTH: 4 212. TYPE: PRT <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Artificial Sequence 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <223> OTHER INFORMATION: Caspase 8 Cleavage Site <4 OOs, SEQUENCE: 30 Ile Glu Thir Asp 1.

<210s, SEQ ID NO 31 &211s LENGTH: 393 212. TYPE: PRT <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <223> OTHER INFORMATION: BACE V4 O- S432 fragment <4 OOs, SEQUENCE: 31 US 7,732,183 B2 67 68

- Continued

Wall Glu Met Wall Asp Asn Lell Arg Gly Lys Ser Gly Glin Gly Tyr 15

Wall Glu Met Thir Wall Gly Ser Pro Pro Glin Thir Lell Asn Ile Luell Wall 2O 25

Asp Thir Gly Ser Ser Asn Phe Ala Wall Gly Ala Ala Pro His Pro Phe 35 4 O 45

Lell His Arg Tyr Glin Arg Glin Luell Ser Ser Thir Arg Asp Luell SO 55 6 O

Arg Gly Wall Tyr Wall Pro Thir Glin Gly Trp Glu Gly Glu 65 70

Lell Gly Thir Asp Lell Wall Ser Ile Pro His Gly Pro Asn Wall Thir Wall 85 90 95

Arg Ala Asn Ile Ala Ala Ile Thir Glu Ser Asp Phe Phe Ile Asn 105 11 O

Gly Ser Asn Trp Glu Gly Ile Luell Gly Luell Ala Ala Glu Ile Ala 115 12 O 125

Arg Pro Asp Asp Ser Lell Glu Pro Phe Phe Asp Ser Lell Wall Glin 13 O 135 14 O

Thir His Wall Pro Asn Lell Phe Ser Luell Glin Luell Gly Ala Gly Phe 145 150 155 160

Pro Luell Asn Glin Ser Glu Wall Luell Ala Ser Wall Gly Gly Ser Met Ile 1.65 17O

Ile Gly Gly Ile Asp His Ser Luell Tyr Thir Gly Ser Lell Trp Thir 18O 185 19 O

Pro Ile Arg Arg Glu Trp Tyr Glu Wall Ile Ile Wall Arg Wall Glu 195

Ile Asn Gly Glin Asp Lell Lys Met Asp Glu Tyr Asn Tyr Asp 21 O 215

Lys Ser Ile Wall Asp Ser Gly Thir Thir Asn Luell Arg Lell Pro Lys 225 23 O 235 24 O

Wall Phe Glu Ala Ala Wall Ser Ile Lys Ala Ala Ser Ser Thir Glu 245 250 255

Phe Pro Asp Gly Phe Trp Luell Gly Glu Glin Lell Wall Cys Trp Glin 26 O 265 27 O

Ala Gly Thir Thir Pro Trp Asn Ile Phe Pro Wall Ile Ser Luell Tyr Luell 27s 285

Met Gly Glu Wall Thir Asn Glin Ser Phe Arg Ile Thir Ile Luell Pro Glin 29 O 295 3 OO

Glin Tyr Luell Arg Pro Wall Glu Asp Wall Ala Thir Ser Glin Asp Asp Cys 3. OS 310 315

Phe Ala Ile Ser Glin Ser Ser Thir Gly Thir Wall Met Gly Ala 3.25 330 335

Wall Ile Met Glu Gly Phe Wall Wall Phe Asp Arg Ala Arg Arg 34 O 345 35. O

Ile Gly Phe Ala Wall Ser Ala Cys His Wall His Asp Glu Phe Thir 355 360 365

Ala Ala Wall Glu Gly Pro Phe Wall Thir Luell Asp Met Glu Asp Gly 37 O 375 38O

Tyr Asn Ile Pro Glin Thir Asp Glu Ser 385 390

<210s, SEQ ID NO 32 &211s LENGTH: 399 212. TYPE : PRT US 7,732,183 B2 69

- Continued <213> ORGANISM: Homo sapiens 22 Os. FEATURE: <221s NAME/KEY: MISC FEATURE <223> OTHER INFORMATION: HIV Protease treated BACE Asp2-2L-TM-His6 <4 OOs, SEQUENCE: 32 Val Glu Met Val Asp Asn Lieu. Arg Gly Lys Ser Gly Glin Gly Tyr Tyr 1. 5 1O 15 Val Glu Met Thr Val Gly Ser Pro Pro Gln Thr Lieu. Asn Ile Leu Val 2O 25 3O Asp Thr Gly Ser Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe 35 4 O 45 Lieu. His Arg Tyr Tyr Glin Arg Glin Lieu. Ser Ser Thr Tyr Arg Asp Lieu. SO 55 6 O Arg Lys Gly Val Tyr Val Pro Tyr Thr Glin Gly Lys Trp Glu Gly Glu 65 70 7s 8O Lieu. Gly Thr Asp Lieu Val Ser Ile Pro His Gly Pro Asn Val Thr Val 85 90 95 Arg Ala Asn. Ile Ala Ala Ile Thr Glu Ser Asp Llys Phe Phe Ile Asn 1OO 105 11 O Gly Ser Asn Trp Glu Gly Ile Lieu. Gly Lieu Ala Tyr Ala Glu Ile Ala 115 12 O 125 Arg Pro Asp Asp Ser Lieu. Glu Pro Phe Phe Asp Ser Lieu Val Lys Glin 13 O 135 14 O Thr His Val Pro Asn Lieu Phe Ser Leu Glin Lieu. Cys Gly Ala Gly Phe 145 150 155 160 Pro Leu. Asn Glin Ser Glu Val Lieu Ala Ser Val Gly Gly Ser Met Ile 1.65 17O 17s Ile Gly Gly Ile Asp His Ser Leu Tyr Thr Gly Ser Lieu. Trp Tyr Thr 18O 185 19 O Pro Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu 195 2OO 2O5 Ile Asin Gly Glin Asp Lieu Lys Met Asp Cys Lys Glu Tyr Asn Tyr Asp 21 O 215 22O Llys Ser Ile Val Asp Ser Gly. Thir Thr Asn Lieu. Arg Lieu Pro Llys Llys 225 23 O 235 24 O Val Phe Glu Ala Ala Wall Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu 245 250 255 Llys Phe Pro Asp Gly Phe Trp Leu Gly Glu Gln Leu Val Cys Trp Glin 26 O 265 27 O Ala Gly. Thir Thr Pro Trp Asn Ile Phe Pro Val Ile Ser Lieu. Tyr Lieu. 27s 28O 285 Met Gly Glu Val Thr Asn Glin Ser Phe Arg Ile Thr Ile Leu Pro Glin 29 O 295 3 OO Glin Tyr Lieu. Arg Pro Val Glu Asp Wall Ala Thir Ser Glin Asp Asp Cys 3. OS 310 315 32O Tyr Llys Phe Ala Ile Ser Glin Ser Ser Thr Gly Thr Val Met Gly Ala 3.25 330 335 Val Ile Met Glu Gly Phe Tyr Val Val Phe Asp Arg Ala Arg Lys Arg 34 O 345 35. O Ile Gly Phe Ala Val Ser Ala Cys His Val His Asp Glu Phe Arg Thr 355 360 365 Ala Ala Val Glu Gly Pro Phe Val Thir Lieu. Asp Met Glu Asp Cys Gly 37 O 375 38O US 7,732,183 B2 71 72

- Continued Tyr Asn Ile Pro Gln Thr Asp Glu Ser His His His His His His 385 390 395

SEQ ID NO 33 LENGTH: 269 TYPE : PRT ORGANISM: Homo sapiens FEATURE: NAME/KEY: MISC FEATURE OTHER INFORMATION: BACE Aa164 - Ser 432 fragment

SEQUENCE: 33

Ala Glu Ile Ala Arg Pro Asp Asp Ser Luell Glu Pro Phe Phe Asp Ser 1. 5 15

Lell Wall Lys Glin Thir His Wall Pro Asn Luell Phe Ser Lell Glin Luell 25

Gly Ala Gly Phe Pro Lell Asn Glin Ser Glu Wall Lell Ala Ser Wall Gly 35 4 O 45

Gly Ser Met Ile Ile Gly Gly Ile Asp His Ser Lell Thir Gly Ser SO 55 6 O

Lell Trp Thir Pro Ile Arg Arg Glu Trp Tyr Glu Wall Ile Ile 65 70 7s 8O

Wall Arg Wall Glu Ile Asn Gly Glin Asp Luell Met Asp Lys Glu 85 90 95

Asn Asp Lys Ser Ile Wall Asp Ser Thir Thir Asn Luell Arg 105 11 O

Lell Pro Lys Wall Phe Glu Ala Ala Wall Ser Ile Ala Ala 115 12 O 125

Ser Ser Thir Glu Lys Phe Pro Asp Gly Phe Trp Lell Gly Glu Glin Luell 13 O 135 14 O

Wall Trp Glin Ala Gly Thir Thir Pro Trp ASn Ile Phe Pro Wall Ile 145 150 155 160

Ser Luell Luell Met Gly Glu Wall Thir Asn Glin Ser Phe Arg Ile Thir 1.65 17O 17s

Ile Luell Pro Glin Glin Tyr Lell Arg Pro Wall Glu Asp Wall Ala Thir Ser 18O 185 19 O

Glin Asp Asp Tyr Phe Ala Ile Ser Glin Ser Ser Thir Gly Thir 195

Wall Met Gly Ala Wall Ile Met Glu Gly Phe Wall Wall Phe Asp Arg 21 O 215 22O

Ala Arg Arg Ile Gly Phe Ala Wall Ser Ala His Wall His Asp 225 23 O 235 24 O

Glu Phe Thir Ala Ala Wall Glu Gly Pro Phe Wall Thir Luell Asp Met 245 250 255

Glu Asp Gly Tyr Asn Ile Pro Glin Thir Asp Glu Ser 26 O 265

We claim: c) diluting the solubilized BACE polypeptide in an aque 1. A method for producing an active recombinant human ous solution having a temperature of about 1°C. to about Beta-site APP cleaving enzyme (BACE) polypeptide com 60 15° C., to obtain a diluted sample; and prising: d) incubating the diluted sample at a temperature of about a) expressing a polynucleotide sequence encoding a BACE polypeptide lacking all or a portion of the sequence 4°C. to about 15° C. until the BACE polypeptide folds corresponding to residues 1-66 of SEQID NO: 1, into an active enzyme. b) solubilizing the BACE polypeptide in a denaturant at a 65 2. The method of claim 1, wherein the BACE polypeptide pH from about 10 to about 11 and in the presence of a lacks a leader sequence and at least a first 24 amino acids of a reducing agent, prosequence. US 7,732,183 B2 73 74 3. The method of claim 1 wherein the polynucleotide 13. The method of claim 8, wherein the polynucleotide sequence encodes a polypeptide that is at least 95% identical sequence expressing the BACE polypeptide is mutated to to the sequence comprising amino acids 66-432 of SEQ ID provide for codons suitable for expression of the BACE NO: 1. polypeptide in a host cell. 4. The method of claim 1 wherein the BACE polypeptide 14. The method of claim 8, wherein the polynucleotide has at least 40% of the activity of recombinant BACE encodes the BACE polypeptide that is truncated by deletion expressed in CHO cells. of all or a portion of a cytoplasmic tail, a prosequence, a 5. The method of claim 3 wherein the BACE polypeptide transmembrane domain, or any combination thereof. has at least 100% of the activity of recombinant BACE 15. The method of claim 9, wherein the polynucleotide expressed in CHO cells. 10 encodes enzymatic cleavage sites to between the coding 6. The method of claim 1, wherein the denaturant is sequence for BACE polypeptide and the coding sequence for selected from the group consisting of urea, guanidine HCI, the N-terminal amino acids. and guanidine thiocyanate, at a concentration from about 6M 16. The method of claim 8, wherein the polynucleotide to about 8M. encodes the BACE polypeptide having an N-terminal amino 7. The method of claim 1, wherein the reducing agents is 15 acid within the protease domain. selected from the group consisting of beta-mercaptoethanol (BME), glutathione, and dithiothreitol (DTT), or a combina 17. The method of claim 16, wherein the BACE polypep tion thereof. tide begins at a position downstream of T' of SEQID NO: 1. 8. A method for producing an active recombinant human 18. The method of claim 17, wherein the BACE polypep BACE polypeptide comprising: tide begins at either R or F of SEQID NO: 1. a) expressing a polynucleotide encoding the BACE 19. The method of claim 10, wherein the polynucleotide polypeptide in a host cell to produce a BACE polypep encodes a soluble BACE polypeptide having an N-terminus at tide inclusion bodies; a position selected from one of residues 24-66 and a C-ter b) solubilizing the BACE polypeptide inclusion bodies to minus at residue S° of SEQID NO: 1. release the recombinant human BACE polypeptide; 25 20. A method for refolding an active recombinant human c) reducing the released BACE polypeptide with a reduc BACE polypeptide comprising: ing agent, a) diluting a solubilized and a reduced BACE polypeptide d) diluting the reduced BACE polypeptide with an aqueous with a low ionic strength aqueous solution having a solution having a temperature of about 1° C. to about 15° temperature of about 1° C. to about 15° C.; C.; and 30 b) incubating the diluted BACE polypeptide at a starting e) incubating the diluted BACE polypeptide at a tempera pH of about 10 to about 11, and at a temperature of about ture from about 4°C. to about 15° C. and at a pH from 1° C. to about 15° C. for about 2 days to about 6 weeks about 10 to about 11, to obtain the active recombinant to allow the BACE polypeptide to fold into an active human BACE polypeptide. enzyme; and 9. The method of claim 8, wherein the polynucleotide 35 c) recovering the active refolded BACE polypeptide. encodes the BACE polypeptide having N-terminal amino 21. The method of claim 20, further comprising applying acids that facilitate expression in a host cell. 10. The method of claim 9 wherein the amino acids com the BACE polypeptide of step c) onto an ion exchange col prise a tag selected from the group consisting of: umn and eluting the BACE polypeptide. (i) a T7 leader sequence or a T7-caspase 8 leader sequence; 40 22. The method of claim 21, further comprising applying (ii) a purification tag; and the eluted BACE polypeptide onto a size exclusion column (iii) the T7 leader sequence or the T7-caspase 8 leader and eluting the BACE polypeptide. sequence and a purification tag. 23. The method of claim 22, further comprising applying 11. The method of claim 10, wherein the nucleotides the eluted BACE polypeptide onto an affinity chromatogra encoding the tag are located at eitheran N-terminus or C-ter 45 phy column comprising an inhibitor, and eluting the active minus of the polynucleotide encoding the BACE polypeptide. recombinant human BACE polypeptide. 12. The method of claim 10, wherein the purification tag is 24. The method of claim 23 wherein the inhibitor is 1-1 of the T7-Tag MASMTGGQQMGR SEQID NO: 8), the six SEQID NO:28. histidine tag, the thioredoxin tag, the hemaglutinin tag, or the GST tag.