US 20060019285A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0019285 A1 Horecka et al. (43) Pub. Date: Jan. 26, 2006

(54) ANALYSIS OF INTRACELLULAR Publication Classification MODIFICATIONS (51) Int. Cl. CI2O I/68 (2006.01) (76) Inventors: Joseph Horecka, Fremont, CA (US); GOIN 33/53 (2006.01) Peter Fung, Sunnyvale, CA (US); (52) U.S. Cl...... 435/6; 435/7.1 Richard M. Eglen, Los Altos, CA (US) (57) ABSTRACT Correspondence Address: Improved methods of determining the intracellular State of a PETERS VERNY JONES & SCHMITT, L.L.P. as well as modifications of the protein are provided 425 SHERMANAVENUE by introducing a Surrogate fusion protein comprising a SUTE 230 member of an enzyme fragment complementation complex and a target protein. After exposing cells transformed with PALO ALTO, CA 94.306 (US) the Surrogate fusion protein to a change in environment, e.g. Appl. No.: 11/170,123 a candidate drug, the cells are lysed, the lysate Separated into (21) fractions or bands, conveniently by and (22) Filed: Jun. 29, 2005 transferring the by Western to a membrane. The bands on the membrane are developed using the other Related U.S. Application Data member of the enzyme fragment complementation complex and a Substrate providing a detectable Signal. The method is (60) Provisional application No. 60/584,709, filed on Jun. found to provide high sensitivity and the ability to observe 30, 2004. modifications of the target protein.

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ANALYSIS OF INTRACELLULAR compound as a drug. Proteins can undergo numerous modi MODIFICATIONS fications, Such as partial or total degradation, , ubiquitination, or dephosphorylation, CROSS-REFERENCE TO RELATED , acylation, , etc. Being able to discern APPLICATIONS the existence of different aspects of the same protein is important in understanding pathways, Such as identifying 0001. This application claims priority from U.S. Provi the major and minor Status of the parent protein and the sional Patent Application No. 60/584,709 filed on Jun. 30, modified protein, where the effect of a candidate compound 2004, which is hereby incorporated by reference in its can be analyzed. Also, by being able to observe patterns of entirety. variations in the protein analogs and partial degradation products, one can identify cellular pathways and their inter REFERENCE TO SEQUENCE LISTING action with other proteins. 0002 Applicants assert that the paper copy of the 0010. There is, therefore, Substantial interest in develop Sequence Listing is identical to the Sequence Listing in ing techniques that will allow the efficient investigation of computer readable form found on the accompanying com cellular proteins, the effect of changes in the cellular envi puter disk. Applicants incorporate the contents of the ronment on the protein, and being able to detect low levels Sequence listing by reference in its entirety. of a protein of interest in the presence of large amounts of BACKGROUND OF THE INVENTION other proteins that may have analogous migration aptitudes. Relevant Literature 0003) 1. Technical Field 0011 U.S. Patent Nos. 6,680,208 and 6,677,128 and 0004. The field of this invention is proteomic assays. WO94/29725 describe various Western blotting techniques. 0005 2. Background PCT/US02/27497 describes the use of fusion proteins employing enzyme fragmentation complexes for determin 0006 AS more is learned about cellular pathways, the interactions of microorganisms with mammals, the growth ing the Status of the fusion protein in a cell. and development of organisms, both prokaryotic and SUMMARY OF THE INVENTION eukaryotic, there is an increasing need for improved meth ods of identifying protein members involved with these 0012. The fate of cellular proteins is determined by various processes. Also, as proteins and their function are employing a genetically modified cell expressing one or identified, there is an increasing interest in being able to more proteins of interest fused to a fragment that is a modulate the activity of proteins associated with diseased member of an enzyme fragment complementation (“EFC) States. By Screening for active compounds that modulate the pair. The lysate of the cell is separated, conveniently activity, one is interested, not only in identifying the effect employing Western blotting, and the fractions or bands of the compound on the target protein, but also any effects developed using the other member of the EFC pair and a the compound may have on other proteins present in the cell Substrate. The observed fractions or bands are limited to or medium. those proteins that comprise the fused fragment, allowing for detection of any form of the protein(s) of interest that 0007. There are numerous ways to separate components retains the fragment. of a complex mixture based on differences in the compo nents of mass, mass-to-charge, conformation, etc. Gel elec trophoresis has been a major methodology for analyzing BRIEF DESCRIPTION OF THE FIGURES mixtures of proteins. Native proteins and denatured proteins 0013 FIG. 1 shows the detection on blots of B-arrestin2 are readily Separated by their migration rate, which for the fusion proteins tagged with both ProLabel and the c-myc most part can be related to their molecular weight. Numer . EAStern blots (upper panels) were photographed ous techniques have been developed for identifying the under white light after incubation with the chromogenic separated protein bands. The was developed to Substrate X-Gal for the indicated times. 1A is an EAStern identify proteins. As part of the Western blot, the proteins are Blot with overnight incubation; 1B is an EAStern Blot with Separated by electrophoresis in a gel, followed by transfer of 2 day incubation; The Western blots (lower panels) were the protein bands to a membrane. The bands are then developed with the chemiluminescent substrate CDP-Star developed by various techniques, Such as labeled antibodies and detected with a CCD camera using the indicated expo or a non-specific protein dye. Other methods of Separation Sure times. Lane 1, prestained MW marker (Invitrogen, Cat. include chromatography, e.g. HPLC, FPLC, capillary elec No. LC5925). Lanes 2-4, lysates of CHO-K1 cells tran trophoresis, etc. siently transfected with plasmids pPL-myc-barr2, pbarr2 0008. The Western blot methodology has many desirable myc-PL, and pEGFP-C1, respectively; 1 C is a Western Blot, properties for identifying proteins in a mixture. However, PAb, 6 min incubation; 1D is a Western Blot, 12 minutes even with 2D protocols, where one is dealing with a lysate incubation; or comparable mixture, there can be protein overlap, diffi 0014 FIG. 2 is a graph showing the amount of IKf8-PL culties in detecting low amounts of a component, inadequate protein present in lysates after HeLa IKf8-PL stable cells Separations, inability to detect modifications of the protein were treated with TNF-C. for the indicated time periods. EFC and the like. values are expressed as percentages of untreated control 0009 Today, one is interested in determining many cells (“0” time point); aspects of the life of a protein in a cell, particularly in 0015 FIG.3 shows EAstern blot detection (upper panel, response to a change in the environment, e.g. a candidate A) of IKf8-PL using the chemiluminescent substrate Beta US 2006/0019285 A1 Jan. 26, 2006

Glo and a 3 min CCD camera exposure. Molecular weight 0024. Because of the convenience of gel electrophoresis marker sizes (kD) are indicated on the left. The anti-actin and Western blotting in allowing one to view the various Western blot (lower panel, B) serves as a loading control. fractions Simultaneously and compare their intensity, West Samples are the same as those described in FIG. 2; ern blotting is the preferred detection System. The membrane of the Western blot permits the simultaneous addition of the 0016 FIG. 4 shows increased CCD camera exposure necessary reagents to the fusion protein and its fragments, So times (in minutes, lower-right corner of each panel, 4A, 5 as to have a comparable concentration of the added reagents. min., 4B 10 min.,4C 15 min., 4D 30 min.) of the EAstern In addition, one may also use antibody detection to compare blot shown in FIG.3. The asterisk (*) in D marks the region the native protein with the fusion protein and its fragments. on the blots where high MW bands appeared with longer Therefore, gel electrophoresis will be described as paradig exposure times, matic of other methods of Separation and isolation. 0017 FIG. 5 shows a bar graph of expression of p53-PL 0025 AS used in this invention, gel electrophoresis and in the presence of varying amounts of siRNA(in specifica Western blotting will be referred to as the EAstern system in tion); emphasizing that the EFC pair consists of an enzyme donor ("ED") and an enzyme acceptor (“EA"). The complexing of 0018 FIG. 6 shows a bar graph of the effect of siRNA on the ED and EA provide the active or holoenzyme. Events cyclin-D1-PL expression (in specification); that result in (1) the expression of the fusion protein or (2) 0019 FIG. 7 shows a bar graph of the effect of siRNA on modification of the fusion protein with a change in migration IKB-PL expression (in specification); and rate of the ED can be measured as an indication of changes of the fusion protein in the cell. 0020 FIG. 8 shows a bar graph of the effect of siRNA on 0026. Any small ED may be used that will allow for STAT-1-PL expression (in specification). detection with an EA and is stable under the conditions of the . For the most part, the ED of B-galactosidase will DESCRIPTION OF THE SPECIFIC be used, but other EDs such as the S-peptide from RNase or EMBODIMENTS the like, can also find use. The ED will generally be at least 0021 Methods and compositions are provided for deter about 35 amino acids and not more than about 100 amino mining the Status of intracellular proteins by employing an acids, generally in the range of about 36 to 90 amino acids, enzyme fragment complementation (“EFC) pair. A geneti preferably below about 75 amino acids. cally modified cell comprising a fusion protein comprising 0027. In some instances it may not be necessary to use the one member of the EFC pair and the protein(s) of interest is entire target protein, Since a portion of the target protein will employed. The cells are lysed and the protein components suffice. Usually at least about 25% of the contiguous amino Separated using any Separation method that allows for indi acids of the protein will be used. In Some instances where a vidual fractions of the different protein components retain protein is processed to Smaller fragments that have physi ing the member of the EFC pair. The use of gel electro ological activity, one may only encode the Smaller fragment. phoresis and Western blotting has been found to be In other instances, one may truncate the protein to use only applicable and advantageous as compared to other methods a functional portion of the protein, e.g. the intracellular of Separation and isolation. The bands comprising the fusion portion of a receptor. protein(s), modified fusion protein(s) and degradation prod ucts containing the fragment are identified, conveniently 0028. After performing whatever changes, if any, in with Western blotting, developed on the membrane using the environment are to be evaluated, the cell is lysed and the other member of the EFC pair and a substrate for the components are separated using 1D or 2D electrophoresis, enzyme. followed by Western blotting. The bands of the Western blot are contacted with an EA and a detectable Substrate, where 0022. The subject method finds particular interest in the enzyme activity is measured from the Signal resulting analyzing cellular pathways and evaluating the effect of a from the enzymatic reaction of the substrate. The amount of change in environment on the cellular pathways involving enzyme product produced is related to the activity of the ED protein(s) of interest. The Subject method is particularly in binding to the EA. The different positions of the bands on useful in evaluating the effect of a change in environment, the membrane indicate the changes in the fusion protein in Such as evaluation of a candidate drug for a specific target the cells. Where one has changed the environment by, for or for the effect on proteins other than the Specific target. It example, the addition of a candidate drug, the changes can finds application in research, high throughput Screening, be related to the presence of the drug. The enzyme activity clinical studies, ADME, and the like. will be influenced by degradation of the fusion protein, 0023 The subject systems employ surrogate fusion pro binding of the fusion protein to a compound complexing teins for determining cellular events, where the Surrogate with the protein of interest, modification of the fusion protein Serves as a measure of the protein of interest. The protein, and the like. The Status of the fusion protein may be System employs various methods and compositions for related to the level of activity of various cellular pathways, determining a cellular event, Such as the Status or State of a where the protein Surrogate being measured is associated protein(s) of interest. Genetic expression constructs are with a pathway. In this way one can measure the activation provided for introducing the genetic construct into a target or inhibition of a pathway, where Such change in the Status cell for expression of the fusion protein. The method relies of the pathway changes the activity and/or nature of the upon the use of EFC pairs, where the holoenzyme is formed fusion protein. by combining the fusion protein or fragment thereof with the 0029. In the electrophoretic gel separation, one may complementary member of the EFC. maintain the native nature of the protein or denature the US 2006/0019285 A1 Jan. 26, 2006

protein using sodium dodecylsulfonate (“SDS”). In the Such as PIPES, MOPS, HEPES, etc., at a concentration in former case, the migration rate will not only depend on size the range of about 50-250 mM, NaCl in the range of about and charge, but also to Some degree on conformation and 250-500 mM, a metal chelating agent, e.g. EGTA, at about interaction with other proteins, e.g. complexes of the fusion 2 to 20 mM, a non-ionic detergent, such as Tween, 5-25 mM protein with other proteins. By contrast, with Separation in Mg Salt and a Stabilizer, Such as Sodium azide at a concen the presence of SDS, most proteins will have the same tration in the range of about 10-20 mM. The pH will be in charge-to-mass ratio and be separated primarily by Size. the range of about 6.5 to 7.5. A conventional buffer has the Either technique may be employed, depending upon the composition: 100 mM PIPES, 400 mM NaCl, 10 mM information one wishes to obtain from the analysis. EGTA, 0.005% Tween, 150 mM NaOH, 10 mM Mg acetate, 0030 Since the migration of the protein will be depen 14.6 mM NaNa, pH 6.9. Following the incubation with EA, dent on size and charge, changes in either or both of these a Substrate is added, there being many convenient fluores will change the migration of the protein. In this way one can cent and chemiluminescent Substrates commercially avail monitor changes in size by degradation, complexation, con able. After sufficient time for the detectable product to form, jugation, Such as glycosylation, ubiquitination, prenylation, the positions of the bands can be visualized and analyzed. isoamide formation, etc., changes in charge as a result of 0035. The sensitivity of the assay is at least about 0.1 ng phosphorylation or dephosphorylation, Similarly , or less in a protein band, 0.09 ng bands having been amidification, acylation, etc. or the opposite, where a group observed. In this way, very small amounts of a modified is removed. One can analyze the band if the nature of the protein may be detected, as well as very Small amounts of band is unknown or where known, the various changes in the the fusion protein. Where the fusion protein undergoes a protein can be readily determined by comparison with variety of modifications, Such as polyubiquitination, poly known proteins. phosphorylation or dephosphorylation, etc., one has the 0031. The changes in the migration of the protein can be opportunity to observe the different forms of the fusion a result of any of a number of processes that result in a size protein and determine the amount of each of the entities. change. Some of these changes may also result in a change in the activity of the ED in forming an active complex with 0036) The fusion protein gene expression construct may EA. In the process of degradation using polyubiquitination, be used initially to determine whether the gene to be inserted a Series of protein products may be formed prior to degra results in a fusion protein that is biologically active to Serve dation by the proteasome. The subject method allows for as a Surrogate for the natural protein. Once it has been shown analysis of these ubiquinated products and an estimate as to that the fusion protein can Serve as a Surrogate, the construct the amount of each of these products. In this way, one may then be used in analyzing the protein under conditions obtains a picture of the process of degradation and its of interest. progress. Alternatively, migration and activity can be modi 0037. The first component of the subject invention is the fied as a result of complex formation between the protein of fusion protein and its expression construct. The ED may be interest as represented by the fusion protein and another at either the C-terminus, the N-terminus or internal to the protein, modification of the fusion protein, partial degrada fusion protein. The particular site of the ED in the fusion tion of the fusion protein, etc. protein will depend upon the ability to include the ED in the 0032. Other methods of separation by size and/or charge, coding Sequence without Significant reduction in the natural Such as chromatography, can also be used. By taking indi activity of the protein of interest, the effect of the position on vidual fractions off of a chromatographic column and adding the modifications of the protein, the nature of the degrada the necessary reagents, any of the ED present can be tion and the ability of the ED to complex with the EA to form determined. Where the individual fractions do not have a an active enzyme. Thus, depending upon how much is known migration rate on the column, the ED containing known about the protein of interest, its structure, site(s) of component may be analyzed. Where the migration rate is binding to other entities, the folding pattern, as to loops, known, one can directly relate the amount of the component B-sheets and C-helices, the manner in which the ED activity in the fraction to the particular form of the fusion protein. will be modulated, e.g. degradation, Steric interference of Use of FPLC is well established as to the packing of the binding with EA by another entity, modification resulting in column and the elution of fractions. changes in conformation or charge, etc., the ED will be 0033. After lysing the cells and, for a Western blotting, Situated to provide the optimized response. For degradation, Separating the proteins by gel electrophoresis, the protein it may or may not matter at what site the ED is situated, this bands are transferred to a membrane in accordance with is also likely to be true in many cases for Steric interference, conventional procedures. Various buffers are available for So long as the fusion protein retains its natural conformation carrying out the transfer and different membranes may be and Susceptibility to degradation. However, for localized used for receiving the bands. Conventional membranes modification, Such as phosphorylation or dephosphorylation, include membranes of , nylon and PVDF. The proteolytic cleavage for maturation, etc., usually it will be particular membrane Selected may vary with the nature of desirable to have the ED in a position that does not influence the proteins of interest and their effect on the assay. The the modification. By knowing the Structure of the protein, bands are then transferred from the gel to the membrane one can Select loops, C-helices, B-sheets, Sites of binding or retaining their position in relation to each other. Once the the like to determine the site for insertion of the ED. transfer is complete, the membrane is placed in an appro 0038. The ED may be inserted into the coding region in priate assay buffer containing EA and the enzyme complex a variety of ways. For a cDNA gene, one may Select a allowed to form. Suitable restriction Site for insertion of the Sequence, where 0034 Buffer is conventional for the complex formation by using overhangs at the restriction site, the orientation is and the assay, generally comprising conventional buffers, provided in the correct direction. Alternatively, one may use US 2006/0019285 A1 Jan. 26, 2006 constructs that have homologous Sequences with the target protein of interest, which gene will not interfere significantly gene and allow for homologous recombination, where the with the transcription of the fusion protein. homologous Sequences that are adjacent in the target gene 0042. It should be understood that the site of integration are separated by the ED in the construct. By using a plasmid of the expression construct will affect the efficiency of in yeast having the cDNA gene, with or without an appro transcription and, therefore, expression of the fusion protein. priate transcriptional and translational regulatory region, one One may optimize the efficiency of expression by Selecting may readily insert the ED construct into the cDNA gene at for cells having a high rate of transcription, one can modify an appropriate Site. Alternatively, one may insert the ED the expression construct by having the expression construct coding region with the appropriate splice Sites in an intron joined to a gene that can be amplified and coamplifies the or in an exon of the gene encoding the protein of interest. In expression construct, e.g. DHFR in the presence of meth this way, one can Select for a site of introduction at any otrexate, or one may use homologous recombination to position in the protein. In Some instances, it will be useful to ensure that the Site of integration provides for efficient make a number of constructs, where the ED is introduced transcription. In this way one may overwhelm the expres into an intron and test the resulting proteins for ED activity Sion of the naturally occurring protein, So that the fusion and retention of function of the protein. Various other protein is the major determinant of the function of the target conventional ways for inserting encoding Sequences into a protein and its modification in the cell. By inserting an gene can be employed. For expression constructs and insertion element, Such as Cre-LOX at a site of efficient decriptions of other conventional manipulative processes, transcription, one can direct the expression construct to the see, e.g., Sambrook, Fritsch & Maniatis, “Molecular Clon Same Site. In any event, one will usually compare the ing: A Laboratory Manual,” Second Edition (1989) Cold enzyme activity from cells in a predetermined environment Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. to cells in the environment being evaluated. One would still (herein “Sambrook et al., 1989”); “DNA Cloning: A Prac retain the naturally occurring protein, which can be analyzed tical Approach,” Volumes I and II (D. N. Glovered. 1985); using labeled antibodies to compare the modifications of the “Oligonucleotide Synthesis” (M. J. Gaited. 1984); "Nucleic naturally occurring protein with the fusion protein. Acid Hybridization'B. D. Hames & S. J. Higgins eds. (1985); “Transcription And Translation'B. D. Hames & S. 0043. Depending upon the purpose of the analysis and J. Higgins, eds. (1984); "Animal Cell Culture"IR. I. Fresh whether in vitro or in Vivo, one may analyze a Substantially ney, ed. (1986); “Immobilized Cells And Enzymes' IRL homogeneous cellular composition or a heterogeneous cel Press, (1986); B. Perbal, “A Practical Guide To Molecular lular composition. That is, one could have a mixture of cells Cloning” (1984). that are free of the fusion protein and contain the fusion protein. AS indicated above, one may wish to analyze the 0.039 The gene encoding the fusion protein will be part modifications of the naturally occurring target protein for of an expression construct. The gene is positioned to be comparison with the fusion protein. Once one has estab under transcriptional and translational regulatory regions lished the relevance of the fusion protein to the effects on the functional in the cellular host. The regulatory region may naturally occurring protein, the comparison need not be include an enhancer, which may provide Such advantages as repeated. limiting the type of cell in which the fusion protein is expressed, requiring Specific conditions for expression, 0044) There are a large number of commercially avail naturally being expressed with the protein of interest, and able transcriptional regulatory regions that may be used and the like. In many instances, the regulatory regions may be the particular Selection will generally not be crucial to the the native regulatory regions of the gene encoding the Success of the Subject invention. Also, the manner in which protein of interest, where the fusion protein may replace the the fusion gene construct is introduced into the host cell will native gene, particularly where the fusion protein is func vary with the purpose for which the fusion gene is being tional as the native protein, may be in addition to the native used. The introduction of the construct may be performed in protein, either integrated in the host cell genome or non vitro or in vivo and will include situations where cells integrated, e.g. on an extrachromosomal element. transformed in culture are then introduced into the mamma lian host. The transcriptional regulatory region may be 0040. In an initial phase of the development of an assay, constitutive or inducible. In the former case, one can have a one may wish to have a Second marker as part of the fusion Steady State concentration of the fusion protein in the host, protein. The Second marker may be any marker that allows while in the latter case one can provide going from the for detection independently of the ED. Such markers include substantially total absence (there is the possibility of leak antigenic that can be recognized by a labeled age) to an increasing amount of the fusion protein until a antibody, polyhistidine that can be detected with a nickel Steady State is reached. With inducible transcription, one can reagent, etc. cycle the cell from a State where the fusion protein is absent to a State where the Steady State concentration of the fusion 0041. In those cells in which the native protein is present protein is present. and expressed, the fusion protein will be competing with the native protein for transcription factors for expression. The 0045 Vectors for introduction of the construct include an Site of the gene in an extrachromosomal element or in the attenuated or defective DNA virus, Such as but not limited chromosome may vary as to transcription level. Therefore, to, herpes simplex virus (HSV), papillomavirus, Epstein in many instances, the transcriptional initiation region will Barr virus (EBV), adenovirus, adeno-associated virus be selected to be operative in the cellular host, but may be (AAV), and the like. Defective viruses, appropriately pack from a virus or other Source that will not significantly aged, which entirely or almost entirely lack viral genes, are compete with the native transcriptional regulatory regions or preferred. Defective virus is not infective after introduction may be associated with a different gene from the gene for the into a cell. Use of defective viral vectors, particularly tropic US 2006/0019285 A1 Jan. 26, 2006

for particular cell types, allows for administration to cells in cell and not active in other cells, e.g. prostate specific a Specific, localized area, without concern that the vector can antigen transcriptional regulatory region for use with proS infect other cells. Thus, a particular locus can be specifically tate cancer cells. targeted with the vector. Specific viral vectors include: a 0050. One may use promoters that are active for a short defective herpes virus 1 (HSV1) vector (Kaplitt et al., 1991, time, Such as viral promoters for early genes, for example, Molec. Cell. Neurosci. 2:320-330); an attenuated adenovirus the human cytomegalovirus (CMV) immediate early pro vector, such as the vector described by Stratford-Perricaudet moter. Other viral promoters include but are not limited to et al. (1992, J. Clin. Invest. 90:626-630 a defective adeno Strong promoters, Such as cytomegaloviral promoters associted virus vector (Samulski et al., 1987, J. Virol. (CMV), SR alpha (Takebe et al., Mole. Cell. Biol. 8:466 61:3096-3101; Samulski et al., 1989, J. Virol. 63:3822 (1988)), SV40 promoters, respiratory syncytial viral pro 3828). moters (RSV), thymine kinase (TK), beta-globin, etc. Alter natively, an inducible promoter can be used. 0046) The vector may be introduced in vitro and in vivo 0051. A large number of promoters have found use in by lipofection. For the past decade, there has been increasing various situations, for various purposes and for various use of lipoSomes for encapsulation and transfection of hosts. Many promoters are commercially available today. nucleic acids in vitro. Synthetic cationic designed to Expression of the fusion protein may be controlled by any limit the difficulties and dangers encountered with lipoSome promoter/enhancer element known in the art, but these mediated transfection can be used to prepare liposomes for regulatory elements must be functional in the host or host in vivo transfection. (Felgner, et. al., 1987, Proc. Natl. Acad. cell selected for expression. Promoters which may be used Sci. U.S.A. 84:7413-7417; see Mackey, et al., 1988, Proc. to control fusion gene expression include, but are not limited Natl. Acad. Sci. U.S.A. 85:8027-8031). The use of cationic to, the SV40 early promoter region (Benoist and Chambon, lipids may promote encapsulation of negatively charged 1981, Nature 290:304-310), the promoter contained in the 3' nucleic acids, and also promote fusion with negatively long terminal repeat of Rous sarcoma virus (Yamamoto, et charged cell membranes (Felgner and Ringold, 1989, Sci al., 1980, Cell 22:787-797), the herpes thymidine kinase ence 337:387-388). Lipofection into the nervous system in promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. vivo has recently been achieved (Holt, et. al., 1990, Neuron 78:1441-1445), the regulatory sequences of the metallothio 4:203-214). The use of lipofection to introduce exogenous nein gene (Brinster et al., 1982, Nature 296:39–42); and the genes into the nervous System in Vivo has certain practical following animal transcriptional control regions, which advantages. Lipids may be chemically coupled to other exhibit tissue specificity and have been utilized in transgenic molecules for the purpose of targeting (see Mackey, et. al., animals: elastase I gene control region which is active in 1988, Supra). Targeted peptides or non-peptide molecules pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; can be coupled to liposomes chemically. Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); 0047. It is also possible to introduce the vector in vitro insulin gene control region which is active in pancreatic beta and in Vivo as a naked DNA plasmid, using calcium phos cells (Hanahan, 1985, Nature 315:115-122), immunoglobu phate precipitation or other known agent. Alternatively, the lin gene control region which is active in lymphoid cells vector containing the gene encoding the fusion protein can (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., be introduced via a DNA vector transporter (see, e.g., Wu et 1985, Nature 318:533-538; Alexander et al., 1987, Mol. al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Cell. Biol. 7:1436-1444), mouse mammary tumor virus Biol. Chem. 263:14621-14624, Hartmut et al., Canadian control region which is active in testicular, breast, lymphoid Patent Application No. 2,012,311, filed Mar. 15, 1990). and mast cells (Leder et al., 1986, Cell 45:485-495), albumin 0.048 Vectors are introduced into the desired host cells in gene control region which is active in liver (Pinkert et al., Vitro by methods known in the art, e.g., transfection, elec 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene troporation, microinjection, transduction, cell fusion, DEAE control region which is active in liver (Krumlaufetal., 1985, dextran, calcium phosphate precipitation, lipofection (lyso Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science Some fusion), use of a gene gun, using a viral vector, with 235:53-58), alpha 1-antitrypsin gene control region which is a DNA vector transporter, and the like. active in the liver (Kelsey et al., 1987, Genes and Devel. 1: 161-171), beta-globin gene control region which is active in 0049 Advantages associated with in vivo introduction of myeloid cells (Mogram et al., 1985, Nature 315:338-340; the fusion protein expression construct are that one has the Kollias et al., 1986, Cell 46:89-94), myelin basic protein expression of the fusion protein in a natural Setting where the gene control region which is active in oligodendrocyte cells factors normally associated with the Status of the cell are in the brain (Readhead et al., 1987; Cell 48:703-712), present. For example, if one were interested in knowing how myosin light chain-2 gene control region which is active in a drug acted on a cell type in relation to the protein of skeletal muscle (Sani, 1985, Nature 314:283-286), prostate interest, by testing the drug in Vivo, one is able to determine Specific antigen control region, which is active in prostate the response of the protein of interest under natural condi cells (U.S. Pat. Nos. 6,197,293 and 6,136,792), and gona tions. Using experimental laboratory animals, one could dotropic releasing hormone gene control region which is isolate cells from the organ or Site of interest and have a active in the hypothalamus (Mason et al., 1986, Science mixture of cells transformed with the fusion protein con 234:1372-1378). Alternatively, expression of the fusion pro Struct and untransformed cells. AS indicated previously, one tein gene can be under control of an inducible promoter, could have a comparison of the naturally occurring target Such as metallothionein promoter, which is induced by protein and the fusion protein. For Specific expression of the exposure to heavy metals or a promoter responsive to fusion protein in a target host cell, one could use a tran tetracycline (tet-responsive promoter). For control of the Scriptional regulatory region that is active in the target host gene transfected into certain brain cells, a glucocorticoid US 2006/0019285 A1 Jan. 26, 2006 inducible promoter can be used, since glucocorticoids can 0055 Kinases are of great significance, such as tyrosine croSS the blood-brain barrier. Alternatively, an estrogen kinases, the MAP kinases, the cyclin dependent kinases, inducible promoter, which would be active in the hypothala GTP kinases, ser/thr kinases, Chk1 and 2, etc. mus and other areas responsive to estrogen, can be used. The 0056 Also of interest are enzyme inhibitors, such as present invention contemplates the use of any promoter C-antitrypsin, antithrombin, cyclophilin inhibitors, protea inducible by a pharmacologic agent that can croSS the Some inhibitors, etc. membrane and for neuronal cells, the blood-brain barrier and influence transcription. 0057. Other proteins of interest are the oncogenes, such as Src, Ras, Neu, Erb, Fos, Kit, Jun, Myc, Myb, Abl, Bcl, etc. 0.052 Vectors containing DNA encoding the following Cytokines, Such as the interferons, C.-Y, interleukins, 1-19, proteins, for example, have been deposited with the Ameri and integrins, adhesins, TNF, receptors, hormones, colony can Type Culture Collection (ATCC) of Rockville, Md. Stimulating factors, growth factors, Such as epidermal Factor VIII (pSP64-VIII, ATCC No. 39812); a Factor VIII growth factor, fibroblast growth factor, etc., bone morpho analog, "LA", lacking 581 amino acids (pDGR-2, ATCCNo. genetic proteins, developmental proteins, Such as the Hox 53100); t-PA and analogs thereof (see co-pending U.S. proteins, or other proteins binding to or regulating proteins application Ser. No. 882,051); VWF (pMT2-VWF, ATCC binding to homeoboxes, e.g. the hedgehog proteins, base ment membrane proteins, heat shock proteins, proteins con No. 67122); EPO (pRK1-4, ATCC No. 39940; pdBPVM taining Krupple and Kringle Structures chaperonins, calcium MTneo 342-12 (BPV-type vector) ATCC No. 37224); and asSociated proteins, e.g. calmodulin, calcineurin, etc., mem GM-CSF (pCSF-1, ATCC No. 39754). brane channels, transporter proteins, etc. 0053. The vector will include the fusion gene under the 0058 Also of interest are the proteins associated with transcriptional and translational control of a promoter, usu proliferation, Such as the cyclins, cyclin dependent kinases, ally a promoter/enhancer region, optionally a replication p53, RB, etc. initiation region to be replication competent, a marker for Selection, as described above, and may include additional 0059 Neuronal proteins, such as B-amyloid, TNF, prion, features, Such as restriction sites, PCR initiation sites, an APP, transporters, e.g. dopamine transporter, receptors, Such expression construct providing constitutive or inducible as NMDA receptors, AMDA receptors, dopamine receptors, expression of EA, or the like. AS described above, there are channels, etc. numerous vectors available providing for numerous differ 0060 Another class of proteins is the transcription factors ent approaches for the expression of the fusion protein in a and their inhibitors or regulatory proteins, Such as Adr Ace, host. Amt, AP, Atif, Att, Baf, Brn, Btf, CEbp, CJun, CEts, CREB, CF, Chop, DP, E2F, Elk, Gata, Hnif, Iii A-H, Irf, NYY, Otf, 0054) Of the protein categories of interest, transcription NFkB, NF-AT, Oct-1, Pea, Pit, PU, S, SP, Stat, Tef, TFIII, factors, inhibitors, regulatory factors, enzymes, membrane TFIIII, Ubf and Usf, while the inhibitors include Erk, IkB, proteins, Structural proteins, and proteins complexing with LIF, Smad, RANTES, Tdg, etc., as well as other proteins any of these proteins, are of interest. Specific proteins asSociated with pathways that induce transcription factor include enzymes, Such as the hydrolases exemplified by Synthesis, activation or inhibition. amide cleaving peptidases, Such as caspases, thrombin, plasminogen, tissue plasminogen activator, cathepsins, 0061 Another class of proteins that are of interest are the dipeptidyl peptidases, prostate Specific antigen, elastase, Surface membrane proteins, where many of Such proteins are collagenase, eXopeptidases, endopeptidases, aminopepti receptors, Such as G protein coupled receptors, hormone dase, metalloproteinases, including both the Serine/threonine receptors, interleukin receptors, Steroid receptors, transport proteases and the tyrosine proteases, hydrolases Such as ers, etc. These receptors include insulin receptor, glucose acetylcholinesterase, Saccharidases, lipases, acylases, ATP transporter, IL-2, 4, etc. receptor, CRXC4, PPAR, etc. Also, cyclohydrolase, cerebrosidases, ATPase, Sphingomyeli the MHC proteins can be of interest. nases, phosphatases, phosphodiesterases, nucleases, both 0062. In some instances, housekeeping proteins will be of endo- and exonucleases, oxidoreductases, Such as the cyto interest, Such as the proteins involved in the tricarboxylic chrome proteins, the dehydrogenases, Such as NAD depen acid cycle, the Krebs cycle, glycogenesis, etc. dent dehydrogenases, Xanthine dehyrogenase, dihydrooro tate dehydrogenase, aldehyde and alcohol dehydrogenase, 0063 AS indicated previously, the genes of each of these aromatase, the reductases, Such as aldose reductase, HMG proteins may be manipulated in numerous ways to fuse ED CoA reductase, trypanothione reductase, etc., and other with the protein while maintaining the biological activity of oxidoreductases, Such as peroxidases, Such as myeloperoxi the protein and ED. dase, glutathione peroxidase, etc., oxidases, Such as 0064 Various pathways will be of interest associated monoamine oxidase, myeloperoxidases, and other enzymes with the different proteins. Thus, pathways involving Signal within the class, Such as NO Synthase, thioredoxin reduc transduction as a result of ligand binding to a Surface tase, dopamine B-hydroxylase, Superoxide dismutase, noX-1 membrane protein receptor, Vesicle formation and transport, oxygenase, etc.; and other enzymes of other classes, Such as multistage Synthesis of cellular components, proteasomes, the transaminase, GABA transaminase, the Synthases, B-ke peroxisomes, Spindle formation, tubulin assemblage, pro toacyl carrier protein Synthase, thymidylate Synthase, Syn cessing of ingested compounds, e.g. toxins, drugs, etc. thatases, Such as the tRNA Synthatase, trans ferases, Such as enol-pyruvyl transferase, glycinamide 0065 One can also use the subject system to measure the ribonucleotide transformylase, COX-1 and -2, adenosine effectiveness of an antisense or RNAi present in a cell. By deaminase. measuring the amount of fusion protein that is expressed and US 2006/0019285 A1 Jan. 26, 2006 present in the cell, the efficiency of the antisense or RNAi in papilloma virus genome (Lusky et al., 1984, Cell 36:391 preventing expression can be measured. In addition, one can 401) and be carried in cell lines such as C127 mouse cells determine the effect of the reduction of the expression of one as a stable episomal element. Other usable mammalian cell protein on another protein, where the other protein is the lines include HeLa, COS-1 monkey cells, melanoma cell fusion protein. For example, where one is interested in the lines Such as Bowes cells, mouse L-929 cells, mouse mam effect of the absence of expression on a pathway, where the mary tumor cells, 3T3 lines derived from Swiss, Balb-c or fusion protein is downstream from the inhibited protein, one NIH mice, BHK or HAK hamster cell lines and the like. can determine whether there are alternative pathways for the 0070 Cell lines may be modified by knocking out spe expression of the fusion protein. Alternatively, one may cific genes, introducing Specific genes, enhancing or dimin investigate the effect of a change in the transduction of a ishing the expression of a protein or the like. The modifi Signal in one pathway on an alternative pathway involving cation may be transient, as in the case of introduction of the fusion protein or on the modification of the fusion antisense DNA, RNAi, or dsRNA or may be permanent, by protein. The combination of inhibition of one protein while deleting a gene, introducing a gene encoding the antisense measuring the expression level or modification of another mRNA of the target protein, adding a dominant recessive protein is a powerful tool in understanding intracellular gene, or the like. Research animals may be employed of activity, the interactions of different pathways and the effect various Strains, where the Strains are a result of naturally of a change in environment on Such intracellular activity. occurring mutations and breeding or using genetic modifi 0.066 The cells comprising the subject constructs may be cations of embyronic or other cells with a resulting geneti used to identify proteins associated with a pathway of cally modified host, which may be vertebrate, e.g. mamma interest, the effect of a change in environment, Such as the lian, fish, insect, or the like, or non-vertebrate, e.g. presence of a drug or drug candidate, on the presence of the nematode, etc. Knock-out mice are extensively described in protein of interest in the cell, changes in the regulation of the literature. One may use the intact host, tissue from the expression, the effect of inhibiting expression of a protein, intact host or cells from the intact host for the purposes of the regulation by a receptor of a cellular pathway and to that this invention. Illustrative of the development of knockout extent, compounds that affect the transduction of a Signal by and knockin mice are Nozawa, et al., Transplantation 2001, the receptor, the activation or deactivation of cellular path 72:147-55; Ferreira, et al., Blood 2001 98:525-32; Kotani, et ways that affect the complex formation or degradation of the al., Biochem. J. 2001, 357:827-34; Zhou, et al., Int. J. fusion protein, expression level of a protein, related to the Radiat. Biol. 2001, 77:763-72; and Chang, et al., Mol. Cell. rates of formation and degradation, etc. Endocrinol. 2001, 180:39-46, and references cited therein, to provide only a few of the large number of publications 0067 Secreted proteins can be determined while they are concerning genetically modified mice. In addition one may intracellular. Prior to being transported from the Golgi to the use hybridomas, where a first cell having the desired gene(s) Surface membrane, a number of Steps must occur and one is fused with an immortalized cell under conditions where can determine the number of Such molecules in the cell and the chromosomes from the first cell are stably maintained. whether they are complexed with other proteins, e.g. dock The gene(s) could be transcription factors, proteins of inter ing protein. est, e.g. human proteins in a non-human host cell, or provide 0068. The host cells will be selected to provide the for enhanced expression of a protein. necessary transcription factors for expression of the fusion 0071. The ED of B-galactosidase is extensively described protein and the other components for the purposes of the in the patent literature. U.S. Pat. Nos. 4,378,428; 4,708,929; determination. The host cells will also be selected toward 5,037,735; 5,106,950; 5,362,625; 5,464,747; 5,604,091; providing an environment resembling the environment 5,643,734; and PCT application nos. WO96/19732; and being Simulated. In many cases primary cells may be WO98/06648 describe assays using complementation of employed, both those maintained in culture and obtained enzyme fragments. The ED will generally be of at least directly from a patient. However, in many other cases, about 35 amino acids, usually at least about 37 amino acids, established cell lines will be used, Since the cell lines can frequently at least about 40 amino acids, and usually not provide the desired environment and allow for direct com exceed 100 amino acids, more usually not exceed 75 amino parisons between Studies, which comparisons may not be acids. The upper limit is defined by the effect of the size of available when using primary cell lines from patients. the ED on the performance and purpose of the determina tion, the effect on the complementation with the EA, the 0069 Established cell lines, including transformed cell inconvenience of a larger construct, and the like. The lines, are Suitable as hosts. Normal diploid cells, cell Strains minimum size that can be used must provide a Signal that is derived from in vitro culture of primary tissue, as well as observable with the products of the cellular events and that primary explants (including relatively undifferentiated cells can be determined with reasonable Sensitivity. The examples Such as hematopoietic stem cells) are also Suitable. Embry in the Experimental Section will provide guidelines as to onic cells may find use, as well as Stem cells, e.g. hemato how to obtain a fusion protein with the desired sensitivity for poietic Stem cells, neuronal Stem cells, muscle Stem cells, the assay. etc. Candidate cells need not be genotypically deficient in a Selection gene So long as the Selection gene is dominantly 0072 The state of all intracellular proteins can be deter acting. The host cells preferably will be established mam mined in accordance with this invention to the extent that the malian cell lines. For stable integration of vector DNA into fusion protein can Serve as a Surrogate for a protein of chromosomal DNA, and for subsequent amplification of the interest, Since all proteins will be Subject to Some modifi integrated vector DNA, both by conventional methods, CHO cation, e.g. degradation. For the most part, the proteins of (Chinese Hamster Ovary) cells are convenient. Alterna interest will be associated with a health function, Such as the tively, vector DNA may include all or part of the bovine effect of an infectious disease, genetic defect, mutation, US 2006/0019285 A1 Jan. 26, 2006 response to a drug, neoplasia, inflammatory response, etc. 0077. For convenience, kits can be provided that may Thus, the change in the migration rate of the fusion protein include all or Some of the major components of the assayS. will be relevant to a physiological function, usually associ For example, a kit may include an expression construct, by ated with the diagnosis and treatment of mammalian hosts, itself or as part of a vector, e.g. plasmid, Virus, usually although there may be other purposes, Such as investigation attenuated, where the expression construct may include a of pathways. marker, a gene encoding a protein for integration, a repli cation initiation site, and the like. The construct may be 0073. A number of substrates for B-galactosidase are extracellular or provided integrated into the genome of a known, where the product is fluorescent. The common cell. In addition to the expression construct, the kit may Substrates are B-D-galactopyranosyl phenols, Such as fluo include EA, Substrate for B-galactosidase, one or more cell rescein, mono- and di-Susbtituted, o-nitrophenyl-B-D-galac lines or primary cells, a graph of response in relation to the toside, B-methylumbelliferyl-B-D-galactoside, X-gal, amount of ED present, buffers, Separation gels, membranes resorufin-B-D-galactoside, commercially available dioxet for the Western blotting, filter paper, etc. In Some instances anes, e.g.Galacto-Light Plus(E) kits (chemiluminescence) and cells may be engineered to provide a desired environment, chlorophenol red. The di-B-D-galactopyianosylfluorescein, Such as high levels of expression of a protein involved in a and chlorophenol red-B-D-galactopyranoside, or analogous pathway of interest, Such as Surface membrane receptors, Substrates, particularly where the product is inhibited from GPCRS, nuclear receptors, e.g. Steroid receptors, transcrip leaking from the cell, may be used as intracellular markers. tion factors, etc. or may have been mutated, So as to have 0.074. During the determination, the cells are maintained reduced levels of expression affecting the expression of the in a viable State, where the cells may be dividing or not fusion protein and one is interested in enhancing the level of dividing. The viable State may be referred to as growing. expression. 0075. The simplest procedure to describe is the use of 0078. The system is initially used to determine whether cells in culture and analysis of the lysate. In this case, the the gene to be inserted results in a fusion protein that is cells are grown in culture. The fusion protein and other biologically active to Serve as a Surrogate for the natural constructs, as appropriate, may be present in the cell inte protein, either providing the activity of the natural protein or grated into the genome or may be added transiently by the responding to modifications in a parallel way to the natural various methods for introducing DNA into a cell for func protein. The activity of the fusion protein may be determined tional translation. The cells may be in culture or in vivo. by using host cells in which the expression of the natural These methods are amply exemplified in the literature, as protein does not occur. This may be as a result of cells in previously described. By employing a marker with the which both copies of the natural protein have been knocked fusion protein for Selection of cells comprising the construct, out; where antisense RNA or RNAi is added to the host cell Such as antibiotic resistance, development of a detectable that inhibits the natural protein but not the fusion protein, Signal, etc., cells in culture comprising the fusion protein can e.g. as to the non-coded 3'-region or includes the 5'-me be separated from cells in which the construct is absent. thionine codon; where antisense RNA or RNAi inhibits a Once the fusion protein is being expressed, the environment transcription factor necessary for expression of the natural of the cells may be modified, if desired. Candidate com protein, where the fusion protein has a different transcrip pounds may be added, ligand for receptors, Surface mem tional regulatory region. brane or nuclear, or the two of these may be added in 0079. In developing the use of the fusion proteins, sys combination, changes in the culture medium may be created, tems can be devised that allow for Screening of genetic other cells may be added for Secretion of factors or binding constructs for their application to the desired investigation. to the transformed cells, viruses may be added, or the like. The user of the System introduces the gene of interest into Given Sufficient time for the environment to take effect the genetic construct provided in the System. By having a and/or taking aliquots of the culture at different time inter multiple cloning Site, the gene is manipulated So as to be vals, the cells may be lysed with a lysis cocktail, the lysis inserted into the multiple cloning site in the correct orien cocktail Subjected to electrophoretic Separation and Western tation and in reading frame with the ED Sequence. Usually, blotting followed by addition of EA and enzyme substrate there will be a linker of not more than 3 codons, preferably and the Signal from the product read. One can then relate this not more than about 2 codons, as a result of the nucleotides result to the amount of fusion protein present, particularly by present in the multiple cloning site remaining between the using Standards where the lysate is Spiked with different ED Sequence and the gene of interest. AS indicated, the amounts of the fusion protein and the amount of active vector that is provided may include the transcriptional fusion protein determined, as well as modification products and/or translational termination Sequences, a polyadenyla of the fusion protein, using Standards for the modified tion Sequence, or other Sequence that encodes a function, product. One would then have a graph relating Signal to e.g. farnesylation, geranylation, etc. Once the fusion protein amount of each of the products of the fusion protein in the construct has been completed, the construct may then be lysate. introduced into the host cell. 0.076 Where the cells are in a viable host, usually the 0080. The steps employed by the subject invention com cells or tissue from the host will be harvested and lysed, so prise: (1) preparing the fusion protein gene and expression that the methodology used for the culture will be the same. construct by insertion of the gene of interest into a multiple If desired, Selection of cells having the construct can be cloning site of a genetic construct provided as part of the achieved by having an antibiotic resistance gene as part of System; (2) introducing the expression construct comprising the construct, So that cells can be Selected using the antibi the fusion protein into a Selected cell host, provided by the otic to avoid dilution of the Sample by cells lacking the System or Selected by the user; (3) incubating the trans COnStruct. formed cell host under conditions that permit expression and US 2006/0019285 A1 Jan. 26, 2006 cell viability; (4) lysing the cells; (5) separating the proteins expresses a 3-arrestin2-myc-PL fusion protein from the using gel electrophoresis; (6) transfer of the separated pro CMV promoter and was constructed in a similar manner. tein bands by Western blotting; (7) adding EA and substrate, First, a Barr2-myc DNA fragment was obtained by PCR usually in two stages to the gel; and (7) identifying the bands amplification from the B-arrestin2 cDNA using a forward on the membrane that are developed by the detectable primer that introduced a BglII site upstream of the Barr2 Start product of the Substrate. When adding EA to the gel, usually codon and a reverse primer that introduced nucleotides a large excess of EA to ED will be added, usually at least encoding the c-myc epitope tag in place of the Barr2 Stop about two-fold excess, frequently at least about five-fold codon followed by an EcoRI site. Second, the amplified excess, and the exceSS may be 20-fold or greater. DNA was digested with BglII and EcoRI and inserted into vector pCMV-PL-C1, which had been prepared by digestion 0081. Using the various components described above for with the same enzymes. The DNA sequences of the cloning use in this invention, the System can be employed to vectors pCMV-PL-N1 and pCMV-PL-C1 are shown in SEQ determine whether the host and expression construct are compatible, whether transient expression, extended expres ID NO:1 and SEQ ID NO:2, respectively. Translation of the Sion, or permanent expression should be employed, whether PL-myc-farr2 and Barr2-myc-PL fusion proteins are shown primary, immortal or cells of intermediate nature should be in SEQ ID NO:3 and SEQ ID NO:4, respectively. The third employed, and the response to known agents. In this way, plasmid used in this experiment was pEGFP-C1 (BD Clon one can optimize the particular System to provide for tech), which expresses untagged green fluorescent protein increased Sensitivity to particular agents. from the CMV promoter and served as a negative control. 0086 Protein blots were prepared from cell lysates using 0082 The system can be used with a data accumulation standard technique. A 10 cm dish of CHO-K1 cells that had and Storage capability, where the data derived from the been grown to confluency was trypsinized and Seeded into System is collected, analyzed and compared to other deter three fresh 10 cm dishes at one-tenth dilution. The cells were minations. In this way, data can be accumulated of the effect then transiently transfected with 5 ug/10 cm dish of the three of various agents on the history of the fusion protein, So that plasmids described above using the FuGENE 6 reagent one can measure how the genetically modified cells respond (Roche). Two-days after transfection, the growth medium to the addition of individual or combinations of candidate was removed and the cells were washed once with 10 ml drugs, variations in rate of change, affect on different path PBS. The cells were then lysed by adding 1 ml of ice-cold ways and the like. By having a database of known responses CLB (Cell Lysis Buffer: 0.5% CHAPS in PBS) and scraping to compounds that have established effects, new candidates cell debris from the plate Surface. The lysates were trans can be compared to Such results for evaluation of their ferred to microtubes on ice and Vortexed periodically over a anticipated physiological effects. Not only can one Study the 30 min period. Samples for gel electrophoresis were pre effect of Such candidates on targets, but also the Side effects pared by combining 0.65 vol cell lysate/0.25 Vol 4x LDS resulting from the presence of Such targets in the media. buffer/0.1 vol 10x reducing agent (the later two components 0.083 AS indicated, the subject method can be used in a obtained from Invitrogen), followed by heating at 70° C. for variety of Situations to great effect, Since the ED is Small 10 min. Electrophoresis of protein Samples on denaturing enough to allow for functioning of the protein of interest as NuPAGE 4-12% Bis-Tris gels and transfer to nitrocellulose a fusion protein with ED, while allowing for ED to complex membranes were carried out according to the manufacturer's with EA to provide a functional enzyme. instructions (Invitrogen). Several replica blots were pre pared at the same time. 0084. The following examples are offered by way of 0087 EAstern blot detection of PL-tagged proteins on illustration and not by way of limitation. one blot was carried out using the chromogenic B-galactosi Experimental dase substrate X-Gal. Following transfer from the gel, the 0085. The first experimental example provides a com blot was equilibrated in EACB (EA Core Buffer, Discov parison of EAstern and Western blot methods for detecting eRx) at room temp. for 5 min, followed by incubating with Specific proteins present in whole-cell lysates. The Specific EAReagent (-0.14 ml/cm membrane, DiscoveRx) at 37°C. proteins in this experiment were fusion proteins tagged with for 1 hr. Without washing, the membrane was placed in a both ProLabel (ED; see WO 03/021265, see SEQ ID NO:1) Petri dish, protein-side up, atop two sheets of 3 MM blotting and the c-myc epitope tag, which allow for EFC and paper saturated with substrate solution (0.33 mg/ml X-Gal, antibody detection methods, respectively. The proteins were 3.5 mM B-mercaptoethanol in EACB). The dish was sealed expressed from plasmids in transiently transfected CHO-K1 with parafilm and placed at room temperature to allow color cells. Plasmid pPL-myc-farr2 expresses a PL-myc-B-arres development. Representative photographs of the EAStern tin2 fusion protein from the CMV promoter and was con blot at various times of development are shown in the upper Structed in two steps. First, a myc-Barr2 DNA fragment was panels of FIG. 1. Specific signal continued to develop over obtained by PCR amplification from a human B-arrestin2 the course of Several days. There was no detectable non cDNA clone (MGC:3754, IMAGE:3028154, Invitrogen) specific signal in the pEGFP-C1 control lane, nor was there using a forward primer that introduced a Bgll Site and background staining of the blot itself (FIG. 1). nucleotides encoding the c-myc epitope tag (amino acids 0088 Western blotting was performed to detect myc EQKLISEEDL; SEQ ID NO: 5) in place of the Barr2 start epitope tagged proteins. Blots were first incubated with codon and a reverse primer that introduced an EcoRI site shaking for 30 min in blocking buffer (TTBS/3% nonfat downstream of the Barr2 Stop codon. Second, the amplified milk powder; TTBS is 100 mM Tris pH 7.5, 0.9% w/v NaCl, DNA was digested with BglII and EcoRI and inserted into 0.1% v/v Tween 20). The blots were then incubated for the vector pCMV-PL-N1, which had been prepared by 45min with antibodies diluted in blocking buffer as follows: digestion with the same enzymes. Plasmid pbarr2-myc-PL anti-myc mouse monoclonal (clone 9E10, Abcam Ltd., Cat. US 2006/0019285 A1 Jan. 26, 2006

No. ab32), 1:5000; anti-myc rabbit polyclonal (Abcam Ltd., blot technique allows for detection levels that exceed those Cat. No. ab9106), 1:1000. The blots were washed four times of traditional Western blotting. for 5 min in TTBS before incubating for 30 min with the 0092. In the following, a series of experiments were corresponding alkaline-phosphatase conjugated Secondary performed varying the parameters of the EAStern protocol in antibodies (Applied Biosystems Cat. Nos. AC32ML and AC31RL) diluted 1:10,000 (v/v) in TTBS/3% NFM. The order to identify conditions that would yield optimal results. membranes were washed as above, followed by a final wash 0093. For the next series of experiments generation of in CSB (CDP-Star Buffer: 100 mM 2-amino-2-methyl-1- cell lysates, protein blot transfer and EAStern analysis of propanol pH 9.5, 0.8 mM MgCl). For development, mem PL-tagged proteins was carried out as described above with branes were incubated at 37 C. for 10 min in substrate the following modifications. First, transfections of HeLa solution (50 uM CDP-Star, 0.5 mg/ml Sapphire II, both from cells with different ProLabel expressing plasmid DNAS (0.5 Applied Biosystems, diluted in CSB), sandwiched between ug) and/or siRNAS to STAT1 (GenBank accession # clear plastic sheets, and their luminescence detected with a NM 032612), Cyclin D1(GenBank accession # CCD camera using various exposure times. Representative NM 003210), p53 (GenBank accession # NM 001641) exposures for the anti-myc monoclonal and polyclonal and IKf8 (GenBank accession # NM 010546) were done in Western blots are shown in the lower panels of FIG. 1. 12 well plates. Second, transfections using Lipofectamine 2000 (Invitrogen), were allowed to go for 16 hours after 0089. The second experimental example illustrates the which the cells were treated with trypsin and collected by sensitivity of the EAstern blot method by following the centrifugation in a microfuge tube. Third, the cell pellet was regulated degradation of a PL-tagged cellular protein, IKB then washed 1x with PBS, centrifuged again and resus PL. In most cells, the binding of inhibitor protein IKB holds pended in 400 mL of DiscoveRx cell lysis buffer (0.5% the transcription factor NF-kB in an inactive form. Stimu CHAPS in PBS+1x COMPLETE protease inhibitor cocktail lation of cells with cytokines, such as TNF-C, activates a {Roche, catalog # 1873580}. The cells were lysed by pathway that leads to phosphorylation, ubiquitination, and continuous vortexing at 37 C. for 30 minutes. Preparation Subsequent degradation of IKB, which in turn leads to the of samples for SDS-PAGE was performed as described release and activation of NF-kB. The regulated degradation above. Samples were run on a NuPAGE 4-12% Bis-Tris gel of IKB is thus an indicator for NF-kB pathway activation. under denaturing conditions and transferred to nitrocellulose 0090 Cell lysate samples for EAStern blot analysis were membrane according to the protocol outlined in the manu prepared from a HeLa/IKB-PL stable cell line (WO facturers instructions (Invitrogen). The membrane was 03/021265, filed Aug. 27, 2002) that was cultured and transferred to a plastic dish and washed 2 times for 5 minutes stimulated with TNF-C. for increasing time periods. A frac in EA core buffer (DiscoveRx). The blot was then incubated tion of each lysate was tested by homogenous EFC assay to with 4 mL of EA solution (DiscoveRx.) at various concen confirm the TNF-C. induced, time-dependent degradation of trations-0.1x, 0.5.x, 1x, 2x and 5x at room temperature on a IKB-PL signal (FIG. 2). Another fraction of each was used rocking platform for two hours. Upon completion of this for gel electrophoresis and transfer to PVDF blots as incubation period, 100 lull of a 20 mg/mL stock of X-gal described above. To detect IKB-PL, the EAstern blot proto (5-bromo-4-chloro-3-indolyl f-D-galactopyranoside) was col was adapted for use with the chemiluminescent Substrate added to the plastic dish and the EA/Substrate mixture was Beta-Glo (Promega Corp., Cat. No. E4720). In particular, incubated at room temperature until bands were detected. after incubating with EA Reagent, the blot was incubated at When using a luminescent Substrate in place of the chro RT for 10 min in a solution of Beta-Glo:EACB (1:1, v/v), magenic Substrate, the incubation period on the blot was 15 Sandwiched between clear plastic sheets, and then its lumi minutes at room temperature and the blot was then exposed nescence detected with a CCD camera. To control for on a UVP EpiChem II darkroom imager for 5-30 minutes to variations in Sample preparation, a replica blot was probed obtain the results. with an anti-actin monoclonal antibody as described above 0094) To demonstrate a correlation between the amount for Western blotting (Abcam Ltd., Cat. No. ab6276); actin of EA used in a reaction and the EFC generated Signal, a levels are not significantly affected by TNF-C. titration of EA amounts was performed. Total cell lysate 0091) A thirty minute exposure of the EAstern blot (FIG. from either HeLa or HeLa cells transfected with STAT-PL or 4D) revealed in the lysates a single band that migrated at the CyclinD1-PL were run on a 4-12% Bis-Tris gel. The predicted molecular weight of IKB-PL (42.1 kDal) and Samples were transferred to nitrocellulose and then Sepa whose abundance throughout the TNF-C. time course paral rately incubated with different concentrations of EA(5x, 2x, leled the EFC Signal in the homogenous assay (compare 1x, 0.5x and 0.1x) for two hours. The blots were then FIGS. 3 and 4). Increased sensitivity was obtained with incubated with X-gal Substrate overnight to produce a chro increased CCD camera exposure time (FIG. 4). Several high magenic Signal. An EFC Signal was detected using EA at a molecular weight bands appeared at the longer exposure concentration as low as 0.5x. There is very low background times (see 10, 15, and 30 min exposures in FIG. 4) that Signal as only ProLabel tagged products produce a Signal on appear to represent IKB-PL modified by poly-ubiquitination, the membrane. Two different ProLabel tagged genes (STAT1 based on the timing of their appearance relative to TNF-C. and Cyclin D1) were used to transfect into HeLa cells. The stimulation and IKB-PL degradation. Poly-ubiquitination is control lanes used in each condition were HeLa cells trans a known post-translational modification that occurs in fected with the pCMV-PL plasmid alone. ProLabel response to cytokine Stimulation and is a prerequisite for expressed in mammalian cells by itself is degraded, while IKB degradation. These bands in Similar experiments using ProLabel tagging a protein results in a stable product that the Western blot method with several commercial prepara can be detected by EFC. tions of anti-IKB antibodies were not previously observed by 0095 Equivalent amounts of total protein were run in us. Thus, the high Specificity and Sensitivity of the EAStern triplicate on a 4-12% Bis-Tris gel. A Western transfer was US 2006/0019285 A1 Jan. 26, 2006

performed as described above. The nitrocellulose mem branes were incubated with different concentrations of EA TABLE 1. Solution for 2 hours at room temperature and then X-gal Substrate was added and the blots were incubated overnight. summarizes data from FIG. 5: 0096) RNA interference (RNAi) technology has become PS3 siRNA Pool a commonly used method to examine the Specific gene Silencing in different cell types. The proceSS involves either SiRNA (nM/well) R1 R2 R3 Avg Small hairpin RNAs (shRNAS) generated by plasmid expres O 12810 15197 17114 15040 O.O1 17836 18116 14348 16767 Sion vectors or chemically Synthesized antisense oligos to O1 5082 3757 3OO2 3947 targeted genes being introduced into cells via transfection. 1.11 1341 1029 796 1055 The interfering RNA binds to the complementary Sequence 11 314 321 294 310 of the expressed gene of interest which causes a cascade of 0.01 Neg Ctrl 196OO 13252 97.01 14184 events leading to the eventual degradation of the target 11 Neg Ctrl 106OO 11987 10241 10943 mRNA and Silencing of gene expression. To analyze the Hela only 190 219 214 208 effects of RNAi introduced into a cell, two methods are commonly used: PCR to monitor mRNA levels or standard Western analysis and antibody detection. Both are well 0099. The next experiment was testing an siRNA pool to established procedures, but they have their drawbacks. PCR Cyclin D1 (Genbank accession # NM 053056). HeLa cells is only a monitor of mRNA levels and cannot account for were co-transfected with Cyclin D1-PL plasmid DNA (0.5 translational modifications that can affect the expression and ug?well) and increasing amounts (1.11-100 nM/well) of function of the gene of interest. Western analysis is limited siRNA to Cyclin D1 (Dharmacon, cat. # M-003210). The to the specificity of the antibody to detect the protein of procedure for transfecting, collecting and assaying the cells interest. The following Sets of experiments demonstrate the was carried out as described above. As seen in FIG. 6, application of EAStern analysis to measure the expression of introduction of the siRNA pool to Cyclin D1 at 1.11 nM different ProLabel tagged genes subjected to RNAi technol generated almost a 90% decrease in the EFC signal. The ogy. A comparison of the results obtained by EAStern and negative control siRNA (same as used in previous experi Western analysis is shown. ment) did not dramatically reduce the EFC activity of Cyclin 0097. In this first experiment, a pool of siRNAS specific D1-PL. A Western was performed on the lysate material for p53 and p53-PL plasmid DNA (DiscoveRx) was used to probing the blot with C.-Cyclin D1 (Upstate, cat # 60-068) co-transfect HeLa cells. The p53 siRNA pool contained an and B-Actin antibodies (AbCam cat # AB6276-100) as a equivalent mixture of four oligos that were specific and control for protein loading. The Western analysis showed a complementary to p53 RNA sequences (Dharmacon, cat if loss of the detected Cyclin D1-PL band (~41 kDa). The Q-003946-00-09). The negative control siRNA is a nonsense detected signal titration with the Cyclin D1 siRNA was not scrambled oligo sequence (Dharmacon, cat # D-001206-13 as evident in the Western. However, with the EAStern 05) which was also tested at Similar concentrations (11 and analysis, a distinct Cyclin D1-PL band was observed and a 0.11 nM). The cells were collected 16 hours post transfec titration of this signal down to 11 nM/well was observed. tion, resuspended in 500 uL of cell lysis buffer (DiscoveRx) and incubated at 37° C. for 30 minutes. To test for EFC 0100. As shown in FIG. 6, SHeLa Cells were co-trans activity of the different conditions, 30 till of a 1:10 dilution fected with CyclinD1 carboxyl-terminally tagged with Pro of the cell lysate from each test condition was incubated with Label and increasing amounts of an SiRNA pool to Cyclin 20 u, of 2x EA for 15 minutes at room temperature in a 384 D1 (Dharmacon, cat. # M-003210) using Lipofectamine well plate. To this reaction, 30 lull of chemiluminescent 2000 reagent (Invitrogen). EFC activity was measured by substrate (DiscoveRX/ABI, Galacton star and Emerald II testing a 1:10 dilution of the lysate. Western and EAstern enhancer mixed together according to Supplier's protocol) analysis were performed to monitor the levels of Cyclin was added and the plate was read after 30 minutes incuba D1-PL protein expressed after titrating the amount of the tion at room temperature. The results as shown in FIG. 5 show that EFC activity of p53-PL is reduced (as much as Cyclin D1 siRNA pool. The EAstern and Western data 98% at the highest concentration) when increasing concen support the EFC results. The EAstern results had a very trations of p53 siRNA is introduced into the cell. The specific band of expected size for Cyclin D1-PL that was negative control siRNA had minimal effect on p53-PL detected by the EA/X-gal substrate that titrated with the expression as measured by EFC activity. concentration of the siRNA tested. The Western blot pro duced a band of interest, but the intensity and titration of the 0098. As shown in FIG. 5, Hela Cells were co-trans detected band did not follow the titration of the siRNA fected with p53-PL (0.5ug?well) and increasing amounts of treatment as well as the EAStern. the siRNA pool to p53 (0.01-11 nM/well) described above. The procedure was performed as described above. (A) EFC TABLE 2 activity demonstrated the effect of the siRNA reducing the expression of the ProLabel tagged p53 gene. (B) Western summarizes data from FIG. 6: and EAStern analysis was performed to monitor the levels of p53-PL protein expressed after titrating the amount of the Cyclin O1 siRNA pool p53 siRNA pool. The results of the EAstern and Western Normalize were in agreement with the EFC data. The EAStern results R1 R2 R3 R4 Avg to 0 cond. had a very specific band that was detected by the EA/X-gal O 548368 S38663 52O175 528248 53.3864 1OO substrate, while the Western had higher levels of non 1.11 408751 451472 44O143 451.496 437966 82 Specific binding/background on the blot. US 2006/0019285 A1 Jan. 26, 2006

(0.01-100 nM/well). The procedure was performed as TABLE 2-continued described above. EFC activity demonstrated the effect of the siRNA reducing the expression of the ProLabel tagged IKE summarizes data from FIG. 6: gene (A). Western and EAStern analysis was performed to Cyclin O1 siRNA pool monitor the levels of IKf8-PL protein expressed after titrating the amount of the IKB siRNA pool (B). The EAstern and Normalize Western data Support the EFC results showing a reduction in R1 R2 R3 R4 Avg to 0 cond. the detected levels of IKf8-PL. 3.33 196283 217857 211138 193343 204655 38 11 61112 65452 67054 63796 64354 12 0103) In this example, the STAT1 gene (GenBank acces 33 36O13 40O26 40047 38053 38535 7 sion # NM 032612) was tagged with ProLabel. This plas 1OO 27205 30272 28687 27471 284.09 5 mid construct (0.5 lug/well) was co-transfected into HeLa neg 466152 508028 512106 S2O090 SO1594 94 cells titrating the amount of either a pool of 4 different ctrl 1.11 neg 478612 S17786 539885 478485 SO3692 94 specific individual siRNAS to STAT 1 (Dharmacon cat. ctrl 11 #M-004718-00-50) at equilivalent concentration or each neg 3946OS 429OO2 441383 40629O 41782O 78 individual siRNA to STAT1 at that same concentration. The ctr 100 negative control SiRNA is a nonsense Scrambled oligo Hela only 2764 2925 2903 2790 2846 1. sequence (Dharmacon, cat # D-001206-13-05) which was also tested at similar concentrations (11, 1.11, 0.11 nM). The cells were collected 16 hours post transfection, resuspended 0101 The next experiment performed was testing a in 500 till of cell lysis buffer (DiscoveRx) and incubated at siRNA pool specific to IKf8 (GenBank accession # 37 C. for 30 minutes. As shown in FIG. 8, co-transfection NM 010546). As done in the previous experiments, IKf8-PL of STAT1-PL plasmid DNA along with increasing concen plasmid (0.5 ug/well) was co-transfected with increasing trations of the pool and individual STAT1 siRNAs (except concentrations of a siRNA pool specific to IKf8 (Dharmacon, for individual siRNA #3) resulted in a reduction of greater cat # M-004765) as well as non-specific negative control than 90% EFC activity as measured by monitoring STAT1 pool siRNA (Dharmacon, cat # D-001206-13-01). A portion PL activity. The negative control scrambled siRNA never of the transfected cell lysate was tested for EFC activity. A had greater than 25% reduction in EFC of the STAT1-PL. dose dependent response to the IKB Specific SiRNA pool was These results were also confirmed by Western analysis using observed. The data Suggests that the even at the highest an C-STAT1 antibody (Cell Signaling Technologies, Cat if concentrations of the siRNA pool, there is only ~62% 9176) and EAstern analysis. The endogenous STAT1 as well reduction in EFC activity (FIG. 7). This may be due to the as the ProLabeled tagged STAT1 were detected by the 16 hour post transfection incubation period with the siRNA Western analysis. The EAstern detected a single distinct and that a longer incubation would result in a larger Silenc band for STAT1-PL. The trend of seeing a reduction or loss ing of the IKB mRNA. The Western and Eastern blots both of STAT1-PL detection based on the individual siRNA show a decrease in detected Signal for IKB-PL that is similar treatments was displayed. The individual STAT1 siRNA #3 to the results seen with the EFC data. The EAStern detected did not affect expression compared to the other individual two bands for IKf8-PL, which could represent constitutively siRNAs that had over 70% reduction in EFC activity at 0.11 expressed and phosphorylated forms of the protein. With the nM/well. This was also shown by the Western and EAstern Western analysis, both the endogenous as well as the Pro analysis. Label tagged forms of IkB were detected. The background signal on the Western blot was higher than that observed for 0104 Lysates from HeLa cells co-transfected with the EAStern analysis. STAT1-PL and three different concentrations of the pool and individual STAT1 siRNAS as well as negative control siR NAS were assayed for EFC activity in a 384 well plate format following the protocol described above. A portion of Results from FIG. 7 are summarized in Table 3 below. the lysates was also run on 4-12% Bis-Tris gels, and Subjected to Western and EAStern analysis, again following IkB siRNA pool the procedure listed above. Normalize R1 R2 R3 R4 Avg to 0 cond. O 529OO1 518077 497526 475631 505059 1OO 1.11 433412 46.4057 451296 483546 458O78 91 The results from FIG. 8 are summarized in Table 4 below: 3.33 442236 430682 415736 4O7296 423988 84 11 4O2944 38O830 3704O9 38.0952 383,784 76 O O.11 1.11 11 33 22O686 22O3OS 222427 232435 223963 44 STAT1 Pool 1OO.O 72.2 6.7 2.8 1OO 19532O 196964. 185139 185962 190846 38 STAT1 #1 1OO.O 27.6 3.8 2.2 neg 681262 726399 745O12 7352O1 721969 143 STAT1 #2 1OO.O 33.9 5.7 3.1 ctrl 1.11 STAT1 #3 1OO.O 108.6 55.0 64.3 neg 689056 721891. 705684 727024 710914 141 STAT1 #4 1OO.O 23.4 3.4 2.8 ctrl 11 Neg Ctrl siRNA 1OO.O 68.2 71.0 86.O neg 614517 682O34 613481 669991 645OO6 128 ctr 100 Hela only 292O 318O 3068 2886 3O14 1. 0105 The following experiments were performed using a bacterially expressed and column purified form of glu 0102) Hela Cells were co-transfected with IKf8-PL(0.5 tathione-S-transferase tagged with ProLabel (GST-PL). pig?well) and increasing amounts of an SiRNA pool to IKf GSTPL was purified to >80% homogeneity as determined US 2006/0019285 A1 Jan. 26, 2006

by Commassie brilliant blue Staining and the concentration GST or GST-PL run on SDS-PAGE, the Promega substrate of the material was determined by Spectrophotometric Aso works well with the EAStern protocol in being able to detect absorbance reading. For all experiments, the GST-PL and the PL tagged gene of interest as a distinct band. The amount the GST alone (control) were diluted in a total cell lysate of GST and GST-PL was titrated from 2.5 to 0.09 ng/lane. isolated from 5x10 cells/mL of CHO-K1 mammalian cells. The proteins transferred to nitrocellulose membrane were incubated with ~5 mL of 5x EA solution (DiscoveRx) for 0106. In the first example, the bacterial GST fusion either 2 or 4 hours and then with 3 mL of the Promega expression constructs: pGEX-6P1 (Amersham) and pGEX substrate for 15 minutes. Bands were clearly detected in the 6P-1+ ProLabel were grown to log phase (as determined by GST PL sample lanes by 30 minutes of exposure time on the ODoo reading) at 37° C. and then induced with 0.5 mM UVP imager. The level of detection was 0.09 ng of GST-PL IPTG (final concentration) overnight at 37° C. The cells protein with a 2 or 4 hour preincubation with 5x EA and a were collected and lysed by Sonication, clarified by centrifu 60 minute exposure when using the luminescent Substrate. gation and the Supernatant was retained. This material was incubated with 200 u of C.-GST-Sepharose resin rocking for 0110 Various concentrations of either GST or GST-PL 1 hour at 4 C. The resin was pelleted, washed 3x with (0.09 ng to 2.5 ng/lane) were loaded onto a 4-12% Bis-Tris PBS--1x protease inhibitor cocktail. The GST fusion protein gel. A Standard Western transfer was performed and the was eluted from the resin with 10 mM reduced glutathione samples were then incubated with 5x EA for 2 or 4 hours (made up in 50 mM Tris-pH8.0). Approximately 5 ng of followed by 15 minute incubation with the Promega proto GST or GST-PL fusion protein was run on three 4-12% type Beta-Glo substrate. 30 and 60 minute exposures on the Bis-Tris acrylamide gels. One gel was stained with Coo gel imager were made. A4 hour incubation with EA and then massie brilliant blue stain. The other two gels were subjected the Beta-Glo Substrate with a 60 minute exposure was able to electrotransfer to nitrocellose membrane. One gel was to clearly detect 0.28 ng lane of the GST-PL protein and probed with an anti-GST polyclonal antibody-Western faintly could detect 0.09 ng. analysis. The Western analysis was done using a 1:7500 0111. The next experiment addresses the question if mix dilution of the anti-GST polyclonal antibody (Amersham, ing EA and luminescent SubStrate together would provide a cat...it 27-4577-01), incubated overnight at 4 C. The next Similar detected Signal to that seen by adding each compo day, the blot was washed 3x in PBS+0.01% Tween 20 and nent Separately. This would reduce one incubation period probed with a secondary antibody (Donkey anti-Goat IgG and shorten the protocol by 1-2 hours. GST and GST-PL HRP, 1:10000 dilution, Promega cat. # V805A), washed 3x were run on SDS-PAGE and probed with a 4:1 mixture of 5x with PBS+0.01% Tween 20 and developed following the EA: Promega luminescent Substrate, for one or two hours protocol of the Pierce SuperSignal West dura extended before being exposed on the imager for 30 or 60 minutes. duration substrate (Pierce, cat #34075). The other blot was With a 60 minute exposure, a titration of the GST-PL from used for an EAStern analysis. The EAStern was incubated 2.5 to 0.09 ng can be detected. with 5x EA for 1 hour and then 100 till of a 20 mg/mL stock of X-gal was added to the blot and incubated on a rocker at 0112 GST or GST-PL (0.09 ng to 2.5 ng/lane) samples room temperature. The GST-PL band was detected -3 hours were loaded onto a 4-12% Bis-Tris gel. A standard Western after the addition of the X-gal Substrate. transfer was performed and the Samples were than incubated with a 4:1 mixture of 5x EA: Promega prototype Beta-Glo 0107 Full-length GST-PL product as well as GST alone substrate for 1 or 2 hours. 30 and 60 minute exposures on the and the degradation products were detected by C.-GST gel imager were made. Both the 1 and 2 hour incubation of Western analysis. The EAStern analysis detected a single EA/Substrate with a 60 minute exposure showed detection band of the correct size for GST-PL (-33 kDa). of GST-PL at 0.09 ng per lane. There were very few 0108) Purified GST and GST-PL (5 ng) were diluted in non-specific bands that appeared. No non-Specific back CHO-K1 cell lysate and run on SDS-PAGE. The gel was ground bands were seen in the GST alone or the CHO-K1 either Coomassie brilliant blue stained (A) or transferred to only (CHO) lysate. nitrocellulose membrane for Western (B) or EAstern (C) 0113 To address if detection of an EAStern signal is analysis. The EAstern had a clear single band at 38 kD with dependent upon the type of membrane used, a comparison of GST-PL and no band with GST, the Western had a number nitrocellulose versus polyvinylidene fluoride (PVDF) mem of diffuse bands with GST-PL and the Coomassie brilliant brane was made. Increasing concentrations of GST and blue showed a band at 38 kD with GST-PL and 28 kD with GSTPL were run on SDS-PAGE, and a Western transfer was GST. Blots are shown in the indicated panels in the priority performed to each membrane type. The four Separate blots provisional patent application. were pre-incubated with 5x EA for 1 hour and then incu 0109 To increase the sensitivity of EAstern detection, a bated with the Promega Substrate for 15 minutes. Or, a 4:1 chemiluminescent Substrate, Promega Beta-Glo (catalog if mix of EA and Substrate was made and incubated on the E4740), was tested. The luminescent substrate was substi blots for 1 hour. With a 10 minute exposure, both mem tuted for X-gal in the assay protocol to take advantage of the branes showed Strong bands in which the detected Signal amplifiable enzymatic reaction of EFC activity to increase was titrating with the concentration of protein run. There the detection Sensitivity and possibly reduce the time needed appears to be a more distinct signal with less background on to detect a signal (4-24 hour exposure time). The Promega the PVDF membrane versus the nitrocellulose. Substrate utilizes a luciferin-galactoside Substrate (6-O-?3 0114. Two different incubation periods with EA and galactopyranosyl-luciferin). Upon ProLabel and EA forming Substrate were tested. Also, two different types of mem an active B-galactosidase complex it will cleave the Sub branes were compared, nitrocellulose and PVDF. First 5x Strate to form luciferin and galactose. The luciferin is then EA was mixed (4:1) with the luminescent substrate and utilized in a firefly luciferase reaction to generate light. With added to the two different types of membranes for 1 hour and US 2006/0019285 A1 Jan. 26, 2006

the results were measured over time. The CHO-K1 lysate 0119) It is evident from the above results that the subject used to dilute the GST and GSTPL protein was also run on System allows for following the fate of a protein as part of the gel to demonstrate there was no non-Specific products the natural metabolism, as affected by a change in the generated from the lysate. A 10 minute exposure on the expression of another protein or as affected by a change in imager was used. Second a duplicate Set of membranes were the external or internal environment. The System is particu pre-incubated with 5x EA for 1 hour and then incubated with larly powerful when used in conjunction with the Eastern the luminescent Substrate for 10 minutes and then a 10 method, as allowing for a simple protocol and ready com minute exposure was taken on the imager. The data Suggest parison of a number of different results evaluated under the that similar results were observed with either mixing EA and Substrate or performing a pre-incubation with EA and then Same conditions. The Subject System provides for an analysis adding the Substrate for a short period. However, there of the various ways that a protein may be modified intrac appeared to be a more distinct signal with less background ellularly during its lifetime, providing a Snapshot of the State on the PVDF membrane versus the nitrocellulose. of the protein during the cellular evolution. Where all of the cells are in the same State, then one obtains the protein 0115) To demonstrate the lowest level of detectable pro profile at the time of the analysis. Where the cells are in tein by EAstern analysis, another titration (0.28-0.01 ng) of different States, then one obtains a picture of the history of GST and GST-PL protein was run on SDS-PAGE. Western the protein during the life of the cell. transfer of the gels was carried out onto nitrocellulose membrane. The blots were incubated with 3 mL of either 2x 0120) The EAstern method for detecting the effect of or 5x EA for 1 hour followed by an incubation for 15 changes in environment on the State of an intracellular minutes with 2 mL of Promega Beta-Glo substrate. The blots protein is highly Sensitive and offerS many advantages over were exposed for either 5 or 10 minutes. The data shows that conventional Western analysis. The methodology allows for with either 2x or 5x EA that a distinct GST-PL band is detection of extremely Small amounts of intracellular pro detected at a concentration as low as 0.03 ng. teins carrying the ED label, so that the various forms of the 0116 GST and GST-PL were run on 4-12% Bis-Tris gels, modified target protein can be observed, Such as ubiquiti Western transfer was done and EAStern analysis performed. nation and polyubiquitination, partial degradation products, The membranes were incubated with 3 mL of 2x or 5x EA addition or removal of Small functional groups, Such as reagent for 1 hour and then 2 mL of Promega Beta glo phosphate, acetyl, etc. The method is readily applicable to substrate. The blots were then exposed for either 5 or 10 high throughput Screening, providing Substantially analo minutes. There is a distinct band for GST-PL that is seen gous steps to the Western. The additional step of transform with the 2x EA as low as 0.03 ng. There is more smearing ing a cell with the fusion protein construct can be performed of the sample when 5x EA is used. commercially and Such cells made available for individual 0117. Ahead to head comparison of EAstern and Western proteins or can be readily performed by those of ordinary analysis was performed. Duplicate gels were run with dif skill in the art. The Subject method provides an important ferent amounts (0.03-0.71 ng) of GST and GST-PL. EAstern advance and advantage to the efforts to understand cellular analysis was performed using 5x EA incubated for 1 hour processes, their response to changes in the environment and and then 15 minutes with the luminescent Substrate and 10 the ability to rapidly Screen compounds for physiological minute exposure. A distinct GST-PL band of the correct size effect, providing the opportunity for monitoring changes was detected by EAstern at each of the different concentra over time. tions. The Western was able to detect GST and GSTPL at each of the concentrations. However there was a higher level 0121 All publications and patent applications cited in of non-specific background on the blot. This could be due to this specification are herein incorporated by reference as if the primary and Secondary antibody concentrations used and each individual publication or patent application were spe further analysis would need to be done. cifically and individually indicated to be incorporated by reference. 0118 GST and GSTPL were run on SDS-PAGE and Western transferred to nitrocellulose. EAStern and Western 0.122 Although the foregoing invention has been analysis were performed as described above. In the case of described in Some detail by way of illustration and example the EAStern, the membrane was exposed for 3 minutes under for purposes of clarity of understanding, it will be readily the UVP imager. The Western transfer was exposed for 1 apparent to those of ordinary skill in the art in light of the minute under the UVP imager. The EAstern showed only a teachings of this invention that certain changes and modi single band, while numerous bands were observed with the fications may be made thereto without departing from the Western, including both the 28 kD and the 38 kD bands. Spirit or Scope of the appended claims.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS : 5 <210> SEQ ID NO 1 <211& LENGTH: 4181 &212> TYPE DNA <213> ORGANISM: Artificial Sequence

US 2006/0019285 A1 Jan. 26, 2006 19

-continued

Met Ser Ser Asn. Ser Lieu Ala Val Val Lieu Glin Arg Arg Asp Trp Glu 1 5 10 15 Asn Pro Gly Val Thr Gln Leu Asn Arg Leu Ala Ala His Pro Pro Phe 2O 25 3O Ala Ser Trp Arg Asn. Ser Glu Glu Ala Arg Thr Asp Arg Pro Ser Glin 35 40 45 Glin Leu Arg Ser Lieu. Asn Gly Glu Pro Asp Ser Asp Lieu Glu Gln Lys 5 O 55 60 Lieu. Ile Ser Glu Glu Asp Leu Gly Glu Lys Pro Gly Thr Arg Val Phe 65 70 75 8O Lys Lys Ser Ser Pro Asn. Cys Lys Lieu. Thr Val Tyr Lieu Gly Lys Arg 85 90 95 Asp Phe Val Asp His Lieu. Asp Llys Val Asp Pro Val Asp Gly Val Val 100 105 110 Leu Val Asp Pro Asp Tyr Lieu Lys Asp Arg Lys Val Phe Val Thr Lieu 115 120 125 Thr Cys Ala Phe Arg Tyr Gly Arg Glu Asp Lieu. Asp Wall Leu Gly Lieu 130 135 1 4 0 Ser Phe Arg Lys Asp Leu Phe Ile Ala Thr Tyr Glin Ala Phe Pro Pro 145 15 O 55 160 Val Pro Asn Pro Pro Arg Pro Pro Thr Arg Lieu Glin Asp Arg Lieu Lieu 1.65 170 175 Arg Lys Leu Gly Gln His Ala His Pro Phe Phe Phe Thr Ile Pro Glin 18O 185 19 O Asn Leu Pro Cys Ser Val Thr Leu Gln Pro Gly Pro Glu Asp Thr Gly 195 200 2O5 Lys Ala Cys Gly Val Asp Phe Glu Ile Arg Ala Phe Cys Ala Lys Ser 210 215 220 Leu Glu Glu Lys Ser His Lys Arg Asn. Ser Val Arg Lieu Val Ile Arg 225 230 235 240 Lys Val Glin Phe Ala Pro Glu Lys Pro Gly Pro Gln Pro Ser Ala Glu 245 250 255 Thir Thr Arg His Phe Leu Met Ser Asp Arg Ser Lieu. His Leu Glu Ala 260 265 27 O Ser Lieu. Asp Lys Glu Lieu. Tyr Tyr His Gly Glu Pro Leu Asn. Wall Asn 275 280 285 Val His Val Thr Asn Asn Ser Thr Lys Thr Val Lys Lys Ile Llys Val 29 O 295 3OO Ser Val Arg Glin Tyr Ala Asp Ile Cys Leu Phe Ser Thr Ala Glin Tyr 305 310 315 320 Lys Cys Pro Val Ala Glin Leu Glu Glin Asp Asp Glin Val Ser Pro Ser 325 330 335 Ser Thr Phe Cys Lys Val Tyr Thr Ile Thr Pro Leu Leu Ser Asp Asn 340 345 35 O Arg Glu Lys Arg Gly Lieu Ala Lieu. Asp Gly Lys Lieu Lys His Glu Asp 355 360 365 Thr Asn Lieu Ala Ser Ser Thr Ile Val Lys Glu Gly Ala Asn Lys Glu 370 375 38O Val Leu Gly Ile Leu Val Ser Tyr Arg Val Lys Val Lys Leu Val Val 385 390 395 400 US 2006/0019285 A1 Jan. 26, 2006 2O

-continued Ser Arg Gly Gly Asp Val Ser Val Glu Leu Pro Phe Val Leu Met His 405 410 415 Pro Llys Pro His Asp His Ile Pro Leu Pro Arg Pro Glin Ser Ala Ala 420 425 43 O Pro Glu Thr Asp Val Pro Val Asp Thr Asn Leu Ile Glu Phe Asp Thr 435 4 40 4 45 Asn Tyr Ala Thr Asp Asp Asp Ile Val Phe Glu Asp Phe Ala Arg Lieu 450 455 460 Arg Lieu Lys Gly Met Lys Asp Asp Asp Tyr Asp Asp Gln Lieu. Cys 465 470 475

<210> SEQ ID NO 4 &2 11s LENGTH 492 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 4 Met Gly Glu Lys Pro Gly Thr Arg Val Phe Lys Lys Ser Ser Pro Asn 1 5 10 15 Cys Lys Lieu. Thr Val Tyr Lieu Gly Lys Arg Asp Phe Val Asp His Lieu 2O 25 3O Asp Llys Val Asp Pro Wall Asp Gly Val Val Lieu Val Asp Pro Asp Tyr 35 40 45 Leu Lys Asp Arg Lys Val Phe Val Thr Leu Thr Cys Ala Phe Arg Tyr 5 O 55 60 Gly Arg Glu Asp Leu Asp Wall Leu Gly Lieu Ser Phe Arg Lys Asp Lieu 65 70 75 8O Phe Ile Ala Thr Tyr Glin Ala Phe Pro Pro Val Pro Asn Pro Pro Arg 85 90 95 Pro Pro Thr Arg Lieu Glin Asp Arg Lieu Lieu Arg Lys Lieu Gly Gln His 100 105 110 Ala His Pro Phe Phe Phe Thir Ile Pro Glin Asn Leu Pro Cys Ser Val 115 120 125 Thr Lieu Gln Pro Gly Pro Glu Asp Thr Gly Lys Ala Cys Gly Val Asp 130 135 1 4 0 Phe Glu Ile Arg Ala Phe Cys Ala Lys Ser Lieu Glu Glu Lys Ser His 145 15 O 155 160 Lys Arg Asn. Ser Val Arg Lieu Val Ile Arg Lys Val Glin Phe Ala Pro 1.65 170 175 Glu Lys Pro Gly Pro Glin Pro Ser Ala Glu Thir Thr Arg His Phe Leu 18O 185 19 O Met Ser Asp Arg Ser Lieu. His Leu Glu Ala Ser Lieu. Asp Lys Glu Lieu 195 200 2O5 Tyr Tyr His Gly Glu Pro Leu Asn Val Asn Val His Val Thr Asn Asn 210 215 220 Ser Thr Lys Thr Val Lys Lys Ile Llys Val Ser Val Arg Glin Tyr Ala 225 230 235 240 Asp Ile Cys Leu Phe Ser Thr Ala Glin Tyr Lys Cys Pro Val Ala Glin 245 250 255 Leu Glu Glin Asp Asp Glin Val Ser Pro Ser Ser Thr Phe Cys Lys Val 260 265 27 O Tyr Thr Ile Thr Pro Leu Lleu Ser Asp Asn Arg Glu Lys Arg Gly Lieu 275 280 285 US 2006/0019285 A1 Jan. 26, 2006 21

-continued

Ala Leu Asp Gly Lys Teu Lys His Glu Asp Thir Asn Lieu Ala Ser Ser 29 O 295

Thr Ile Wall Glu Gly Ala Asn Lys Glu Wall Leu Gly Ile Telu Wall 305 310 315 320

Ser Tyr Arg Wall Lys Wall Lys Telu Wal Wall Ser Arg Gly Gly Asp Wall 325 330 335

Ser Wall Glu Leu Pro Phe Wall Telu Met His Pro Pro His Asp His 340 345 35 O

Ile Pro Leu Pro Pro Glin Ser Ala Ala Pro Glu Thr Asp Wall Pro 355 360 365

Wall Asp Thr Asn. Teu Ile Glu Phe Thr Asn Ala Thr Asp Asp 370 375

Asp Ile Val Phe Glu Asp Phe Ala Arg Telu Arg Teu Gly Met Lys 385 390 395 400

Asp Asp Asp Asp Asp Glin Telu Cys Glu Glin Teu Ile Ser Glu 405 410 415

Glu Asp Leu Gly Ile Teu Glin Ser Thr Wall Pro Arg Ala Arg Asp Pro 420 425 43 O

Pro Wall Ala Thr Met Ser Ser Asn. Ser Lieu Ala Wal Wall Telu Glin Arg 435 4 40 4 45

Arg Asp Trp Glu Asn. Pro Gly Wall Thr Glin Teu Asn Arg Telu Ala Ala 450 455 460

His Pro Pro Phe Ala Ser Trp Asn. Ser Glu Glu Ala Arg Thr Asp 465 470 475 480

Arg Pro Ser Glin Glin Teu Ser Lieu Asn Gly Glu 485 490

SEQ ID NO 5 LENGTH 10 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Description of Artificial Sequence: Synthetic c-myc epitope tag

<400 SEQUENCE: 5 Glu Gln Lys Lieu. Ile Ser Glu Glu Asp Lieu 1 5 10

What is claimed is: determining the presence of Said fusion protein and modi 1. A method for determining the State of an intracellular fications thereof in said fractions by means of said ED target protein using a Surrogate protein comprising a fusion binding to Said EA in the presence of Substrate for Said protein of at least a Substantial portion of Said target protein active enzyme. and an ED, wherein said ED is a member of an enzyme 2. A method according to claim 1, wherein Said enzyme is fragment complementation pair that forms an active enzyme B-galactosidase. with the complementary fragment EA, wherein Said method 3. A method according to claim 2, wherein Said ED is less comprises: than about 100 amino acids. 4. A method according to claim 2, wherein Said ED is at lysing a cell transformed with an expression construct of the C-terminus of Said target protein. Said fusion protein resulting in intracellular expression 5. A method according to claim 1, wherein prior to lysing of Said fusion protein; Said cell, Said cell is contacted with an agent to determine the Separating Said lysate into fractions where Said fusion effect of Said agent on the State of Said target protein in Said protein and modifications thereof comprising Said ED cell. are separated by at least one of Size and mass-to-charge 6. A method according to claim 1, wherein Said agent is ratio; and RNAi for a protein other than Said target protein. US 2006/0019285 A1 Jan. 26, 2006 22

7. A method for determining the state of an intracellular Separating Said lysate using gel electrophoresis and West target protein using a Surrogate protein comprising a fusion ern blotting to provide a Western blot; and protein of at least a Substantial portion of Said target protein and an ED, wherein said ED is a member of an enzyme developing Said Western blot using Said EA and a detect fragment complementation pair that forms an active enzyme able Substrate, whereby any fusion protein and modi with the complementary fragment EA, wherein Said method fied fusion protein containing Said ED is detected. comprises: 14. A method according to claim 13, including the addi tional Step of determining the natural target protein on Said lysing a cell transformed with an expression construct of gel. Said fusion protein resulting in intracellular expression 15. A method according to claim 13, wherein said Western of Said fusion protein; blot employs a PVDF membrane. Separating Said lysate using gel electrophoresis and West 16. A method according to claim 13, wherein said Western ern blotting to provide a Western blot; and blot employs a nitrocellulose membrane. developing Said Western blot using Said EA and a detect 17. A method according to claim 13, wherein said ED is able Substrate, whereby any fusion protein and modi of from 35 to 100 amino acids. fied fusion protein containing Said ED is detected. 18. A method according to claim 13 wherein prior to 8. A method according to claim 7, wherein Said enzyme is lysing Said cell, Said cell is contacted with an agent to B-galactosidase. determine the effect of Said agent on the State of Said target 9. A method according to claim 8, wherein said ED is less protein in Said cell. than about 100 amino acids. 19. A method according to claim 18, wherein Said agent 10. A method according to claim 8, wherein said ED is at is a candidate drug. the C-terminus of Said target protein. 11. A method according to claim 7, wherein prior to lysing 20. A method according to claim 18, wherein Said agent Said cell, Said cell is contacted with an agent to determine the is RNAi. effect of Said agent on the State of Said target protein in Said 21. A kit comprising a nucleic acid Sequence encoding a cell. fusion protein comprising a target protein and an ED, EA, a 12. A method according to claim 7, wherein Said agent is substrate for the enzyme formed by the complex of said ED RNAi for a protein other than Said target protein. and EA, and at least one of a gel, membrane, buffer for 13. A method for determining the State of an intracellular Western blotting, and blotting filter paper. target protein using a Surrogate protein comprising a fusion 22. A kit according to claim 21, wherein Said enzyme is protein of at least a Substantial portion of Said target protein B-galactosidase. and an ED, wherein Said ED is a member of B-galactosidase 23. A kit according to claim 22, wherein Said Substrate that forms an active enzyme with the complementary frag produces a fluorescent product. ment EA, wherein Said method comprises: 24. A kit according to claim 22, wherein Said Substrate lysing a mammalian cell transformed with an expression produces a chemiluminescent product. construct of Said fusion protein resulting in intracellular expression of Said fusion protein;