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. U.S. Patent Jun. 8, 2010 Sheet 10 of 15 US 7,732,183 B2 *****************{3#ž**$3&#####33333.3%,g. {3; ; , S: U.S. Patent Jun. 8, 2010 Sheet 11 of 15 US 7,732,183 B2 G. s. U.S. Patent Jun. 8, 2010 Sheet 12 of 15 US 7,732,183 B2 & - s : : * *: s : 8 : 88: ... -- pit-is-(i. S.S. & E is :::cti:388 8: post-BACE (R.S. Parification Steps. U.S. Patent Jun. 8, 2010 Sheet 13 of 15 US 7,732,183 B2 U.S. Patent Jun. 8, 2010 Sheet 15 Of 15 US 7,732,183 B2 : 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.