Abstract:
An expression system for producing and isolating large quantities of protein. The system comprises an expression vector containing a first coding region which codes for glutathione-S-transferase operatively connected to a baculovirus promoter, a second coding region in-frame with the first coding region, and a restriction region downstream of the first coding region, into which the second coding region is inserted. A fusion protein encoded by the first and second coding region is produced by expression of the vector. Examples of this second coding region include Lck, LynB, Syk, Blk, Fyn, and Yes. A process for expression of the vector in a host cell such as  Spodoptera frugiperda  is also included.

Description:
FIELD OF THE INVENTION 
     The present invention relates to processes for expression of proteins and to expression vectors and host cells used therefor. 
     BACKGROUND OF THE INVENTION 
     The lck gene product, p56 lck , is a member of the src family of protein tyrosine kinases. Cooper, J. A. (1990) in  Peptides and Protein Phosphorylation  (Kemps, B. E., ed) pp. 85-113, CRC Press, Boca Raton, Fla. The lck protein is normally expressed in T lymphocytes and natural killer cells, where it likely performs a variety of functions relating to signal transduction through ligand binding to selected surface proteins. Bolen, J. A., and Veillette, A. (1989)  Trends Biochem. Sci . 14, 404-407; Rudd, C. E. (1990)  Immunol. Today  11, 400-406. In T-cells, p56 lck  forms a non-covalent complex with the CD4 and CD8a. Veillette, A., Bookman, M. A., Horak, E. M., and Bolen, J. A. (1988). For this reason, p56 lck  is believed to aid in mediation of signals emanating from the T-cell antigen receptor through ligation of CD4 or CD8 to non-polymorphic determinants on antigen-bearing major histocompatibility molecules. Shaw, A. S., Chalupny, J., Whitney, J. A., Hammond, C., Amrein, K. E., Kavathas, P., Sefton, B. M., and Rose, J. K., (1990)  Mol. Cell. Biol . 10, 1853-1862; Doyle, C., and Strominger, J. L. (1987)  Nature  330, 256-259; Norment, A. M., Salter, R. D., Parham, P., Engelhard, V. H., and Littman, D. R. (1988)  Nature  336, 79-81. More recently, p56 lck  has been implicated as a signaling component of the high affinity interleukin-2 receptor. Hatakeyama, M., Kono, T., Kobayashi, N., Kawahara, A., Levin, S. D., Perlmutter, R. M., and Tanaguchi, T. (1991)  Science  252, 1523-1528. 
     A better understanding of the structure and regulation of p56 lck  and similar proteins would clearly contribute to our knowledge of early signal transduction events and a source of large quantities of purified p56 lck  would be useful. While early analysis of p56 lck  functions have been greatly facilitated by antibodies directed against this protein, immunoaffinity purification has been hampered by lack of an abundant source of enzyme. This difficulty has been addressed in part by baculovirus expression systems. Summers, M. D., and Smith, G. E. (1987).  A Manual for baculovirus vectors and insect cell culture procedures , Texas A&amp;M bulletin No. 1555, (College Station, Texas Agricultural Experimental Station and Texas A&amp;M University), 10-39. Recent studies using a baculovirus expression system have reported significant purification of p56 lck  using conventional chromatography methodologies. Ramer S. E., Winkler, D. G., Carrera, A., Roberts, T. M., and Walsh, C. T. (1991)  Proc. Natl. Acad. Sci. USA  88, 6254-6258; Watts, J. D., Wilson, G. M., Ettehadieh, E., Clark-Lewis, I., Kubanek, C., Astell, C. R., Marth, J. D., and Aebersold, R, (1991)  J. Biol, Chem . 267, 901-907. While this approach results in purified enzyme, multiple column enzyme purification is costly, time-consuming, and requires large amounts of starting material. 
     Glutathione-s-transferase (Gst) is a protein well known to bind to glutathione (Smith, D. B., and Johnson, K. S. (1988)  Gene  67, 31-40). Glutathione resin may be used in column chromatography. The above baculovirus expression systems, however, do not employ Gst. 
     BRIEF DESCRIPTION OF THE INVENTION 
     The present invention relates to processes for expressing isolated forms of proteins and to expression vectors and host cells useful for such processes. In particular, this invention relates to an expression vector, comprising: 
     (a) a first coding region, which codes for a polypeptide capable of binding to gluthathione, operatively connected to a promoter, 
     (b) a second coding region in-frame with the first coding region, and 
     (c) at least one restriction site between the first and second coding regions; 
     wherein a fusion protein of the first and second coding regions would result from expression of the vector. Vectors derived from baculovirus are preferred. 
     Further in accordance with this invention is a host cell comprising such a vector. The preferred host cell is a  Spodoptera frugiperda  cell, particularly an Sf9 cell, although other host cells are suitable (see below). 
     Such vectors and host cells are useful in a process for expressing a protein in isolated form, which comprises: 
     (a) treating such a host cell under conditions allowing expression of the vector, whereby a fusion protein of the first and second coding regions will be expressed; 
     (b) exposing proteins from the host cell to glutathione resin, whereby the fusion protein will adhere to the resin; and 
     (c) cleaving the expression product of the second coding region from the resin-bound fusion protein. 
     Further in accordance with the present invention is a process for expressing a nucleic acid sequence, which comprises: 
     (a) inserting the nucleic acid sequence into a baculovirus expression vector in-frame with a first coding region for a polypeptide capable of binding to glutathione, 
     wherein the coding region is operatively linked to a promoter; 
     (b) placing the vector into a host cell; 
     (c) treating the host cell under conditions allowing expression of the vector, resulting in expression of a fusion protein of the first coding region and the sequence inserted in step (a); 
     (d) exposing proteins from the host cell to glutathione resin, whereby the fusion protein adheres to the resin; and 
     (e) treating the adhered fusion protein with a protease to release the expression product of the nucleic acid sequence from the resin. 
     For the first coding region, the inventors prefer a sequence encoding glutathione-s-transferase (nucleotide SEQ. ID. NO.: 1; amino acid SEQ. ID. NO.: 2) or a fragment thereof capable of binding to glutathione. This system combines the high level expression of foreign proteins with baculovirus vectors (e.g., in Sf9 cells) and the ability of Gst fusion proteins to bind to glutathione resin. Treatment of the glutathione-binding fusion protein with a proteolytic substance such as thrombin can thus liberate the desired protein from the glutathione-binding portion of the fusion protein. The glutathione-binding portion remains bound to the resin, thus purifying the desired protein. 
     This expression system presents advantages over other systems, because it allows the practitioner (1) to produce large quantities of protein, (2) to purify significant amounts of active protein by a single chromatography step, (3) to use a wide range of extraction conditions, including non-denaturing detergents to maintain protein function, (4) to use anti-Gst antibodies, allowing for screening of recombinant baculoviruses that express cloned sequences to which antibodies have not been generated or proteins whose function can not be measured, (5) to use a multiple cloning site with many restriction sites for convenient ligation, and (6) to use and/or study thrombin because it includes a thrombin cleavage site. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The following definitions apply to the terms as used throughout this specification, unless otherwise limited in specific instances. 
     The term “fusion protein” refers to a protein or polypeptide that has an amino acid sequence having portions corresponding to amino acid sequences from two or more proteins. The sequences from two or more proteins may be full or partial (i.e., fragments) of the proteins. Such fusion proteins may also have linking regions of amino acids between the portions corresponding to those of the proteins. Such fusion proteins may be prepared by recombinant methods, wherein the corresponding nucleic acids are joined through treatment with nucleases and ligases and incorporated into an expression vector. Preparation of fusion proteins is generally understood by those having ordinary skill in the art. 
     The phrase “polypeptide capable of binding to glutathione” refers to proteins, protein fragments, and synthetic polypeptides capable of binding to glutathione. Examples include glutathione-s-transferase and fragments thereof. Suitable fragments may be generated by gene amplification using 5′ and 3′ primers before translation or by proteolytic cleavage (see Table 2) after translation. 
     The term “coding region” refers to an open reading frame; i.e., a portion of a nucleic acid that has a sequence that would be translated to form a sequence of amino acids. The term “coding region” includes sequences of naturally occurring proteins as well as sequences resulting from modifications (insertions, deletions, mutations, disruptions) obtained through recombinant methods. 
     The term “linking region” refers to a sequence of amino acids between coding regions from different sources in a fusion protein. Typically, linking regions encode sites recognized by proteases and thus allow the expression products of the coding regions to be separated from each other. 
     The phrase “operatively linked to a promoter” means that the promoter is capable of directing the expression of the associated coding region. Coding regions for the fusion protein may also be operatively linked to other regulatory elements, such as enhancers. 
     The preferred embodiment employs a Gst sequence within commercially available expression vector pGEX-2T. This sequence is derived from  Schistosoma japonicum . A number of species are known to produce active isoforms of Gst, all of which are useful in the present invention. 
     Coding regions for the fusion protein may be spliced into an expression vector by means well understood by those having ordinary skill in the art. Suitable expression vectors may be constructed using standard recombinant DNA techniques known in the art, many of which are described in Sambrook, et al.,  Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spring Harbor Laboratory, Cold Spring Habor, N.Y. (1989). 
     Suitable expression vectors in accordance with the present invention comprise a coding region for a polypeptide capable of binding to glutathione, along with an in-frame sequence for the protein to be isolated. The coding region for the protein to be isolated may be located upstream or downstream of the coding region for the glutathione-binding polypeptide. Preferred are expression vectors comprising one or more regulatory DNA sequences operatively linked to the DNA sequence coding for all or part of Gst. 
     Expression vectors useful in the present invention typically contain an origin of replication, a promoter located 5′ to (i.e., upstream of) the Gst fusion protein sequence, which is followed by downstream transcription termination sequences, and the remaining vector. Control regions derived from a number of sources may be employed in accordance with the present invention. Suitable origins of replication include, for example, the Col E1, the SV40 viral and the M13 orgins of replication. Suitable promoters include, for example, the cytomegalovirus promoter, the lac Z promoter, the gal 10 promoter and the  Autographa californica  multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter. Suitable termination sequences include, for example, SV40, lac Z and AcMNPV polyhedral polyadenylation signals. An expression vector as contemplated by the present invention is at least capable of directing the replication, and preferably the expression, of the nucleic acids encoding the fusion proteins. 
     The expression vectors may also include other DNA sequences known in the art; for example, stability leader sequences which provide for stability of the expression product; secretory leader sequences, which provide for secretion of the expression product; sequences that allow expression of the structural gene to be modulated (e.g., by the presence or absence of nutrients or other inducers in the growth medium); marking sequences, which are capable of providing phenotypic selection in transformed host cells (e.g., genes for neomycin, ampicillin, and hygromycin resistance and the like); and sequences that provide sites for cleavage by restriction endonucleases. All of these materials are known in the art and are commercially available. 
     The characteristics of the actual expression vector used must be compatible with the host cell to be employed. The vector thus may include sequences which allow expression in various types of host cells, including but not limited to prokaryotes, yeasts, fungi, plants and higher eukaryotes. For example, when expressing DNA sequences in a mammalian cell system, the expression vector should contain promoters isolated from the genome of mammalian cells, (e.g., mouse metallothionien promoter), or from viruses that grow in these cells (e.g., baculovirus promoter, vaccinia virus 7.5 K promoter). 
     Suitable commercially available expression vectors into which DNA sequences for the fusion proteins may be inserted include the mammalian expression vectors pcDNAI or pcDNA/Neo, the baculovirus expression vectors pBlueBac and pVL1393 (which is preferred), the prokaryotic expression vector pcDNAII and the yeast expression vector pYes2, all of which may be obtained from Invitrogen Corp., San Diego, Calif. Preferred are commercially available vectors that already have Gst sequences included, such as pGEX-2T. 
     The present invention additionally concerns host cells containing an expression vector that comprises a DNA sequence coding for a Gst fusion protein. The host cells preferably contain an expression vector which comprises all or part of the DNA sequence for the protein to be isolated together with a DNA sequence for a polypeptide capable of binding glutathione. See, for example, the expression vector appearing in the Experimental Procedures hereinbelow, which is preferred. Further preferred are host cells containing an expression vector comprising one or more regulatory DNA sequences capable of directing the replication and/or the expression of and operatively linked to a DNA sequence coding for all or part of the fusion protein. Suitable host cells include both prokaryotic and eukaryotic cells. Suitable prokaryotic host cells include, for example,  E. coli  strains HB101, DH5α, XL1 Blue, Y1090 and JM101. Suitable eukaryotic host cells include, for example,  Spodoptera frugiperda  insect cells (which are preferred), COS-7 cells, human skin fibroblasts, and  Saccharomyces cerevisiae  cells. 
     Expression vectors may be introduced into host cells by various methods known in the art. For example, transfection of host cells with expression vectors can be carried out by the calcium phosphate precipitation method. However, other methods for introducing expression vectors into host cells, for example, electroporation, liposomal fusion, nuclear injection, and viral or phage infection can also be employed. 
     Once an expression vector has been introduced into an appropriate host cell, the host cell may be cultured under conditions permitting expression of large amounts of the fusion protein. 
    
    
     BRIEF DESCRIPTION OF THE FIGURES 
     FIGS.  1 A- 1 B: Construction of pBMS-I 
     A. Outline of the cloning procedure. The glutathione-s-transferase gene was cloned into the Bam H-1 site of the Sf9 expression vector pVL1393 to make the Gst fusion expression vector pBMS-I. The restriction map of the pBMS-I polylinker, and the thrombin cleavage site are shown. The DNA sequence at the bottom of FIG. 1A is designated SEQ ID NO:.5. The amino acid sequence at the bottom of FIG. 1A is designated SEQ ID NO:.6. 
     B. Schematic of the GstLck fusion junction. lck was joined to the Gst coding sequence using a Stu-1 site located 24 base pairs upstream of the lck intiation methionine codon. The DNA sequence at bottom of FIG. 1B is designated SEQ ID NO:.7. The amino acid sequence at the bottom of FIG. 1B is designated SEQ ID NO:.8. 
     FIGS.  2 A- 2 C: Analysis of GstLck purified from Sf9 cells. 
     A. SDS-PAGE analysis and Coomassie staining pattern. Lane 1 shows the result from 50 μg of total protein from infected Sf9 cells; lane 2, 1 μg of purified GstLck; lane 3, 0.5 μg of thrombin-cleaved GstLck (recombinant p56 lck ). 
     B. SDS-PAGE analysis of autophosphorylated GstLck. Lane 1 shows the result from autophosphorylation of GstLck; lane 2, autophosphorylation of recombinant p56 lck . 
     C. Western blot analysis of the sample used in panel B using a polyclonal rabbit anti-lck antibody. Lane 1 shows the result from GstLck; Lane 2, recombinant p56 lck . 
     FIGS.  3 A- 3 B: Autophosphorylation of GstLck. 
     A. Western blot analysis of p56 lck . Lane 1 shows the result from immunoprecipitated p56 lck  from CEM-6 cells; lanes 2-4, GstLck from infected Sf9 cell lysates purified using the following methods. Lane 2, immunoprecipitation using anti-lck polyclonal antibodies; lane 3, immunoprecipitation using anti-Gst polyclonal antibodies; lane 4, affinity purification using glutathione resin. 
     B. Analysis of the enzymatic activity of p56 lck  or GstLck purified as outlined in panel A. Activity was assessed by autophosphorylation. The same protein samples and quantities were loaded as in panel A. 
     FIGS.  4 A- 4 B: Phosphorylation of enolase by GstLck. 
     A. Phosphorylation of enolase as a function of GstLck concentration. Each reaction was carried out for 1 minute at 30° C., with 3 μg of enolase as substrate, and varying amounts of GstLck. Lane 1 shows the result from 0 μg GstLck; Lane 2, 0.04 μg GstLck, lane 3, 0.08 μg GstLck; lane 4, 0.12 μg GstLck; lane 5, 0.2 μg GstLck; lane 6, 0.28 μg GstLck; lane 7, 0.36 μg GstLck; lane 8, 0.44 μg GstLck; lane 9, 0.52 μg GstLck. 
     B. Time course of enolase phosphorylation by GstLck. Each reaction was carried out at 30° C., with 0.4 μg of GstLck, and 3 μg of enolase as substrate. Lane 1 shows the result after 0 minutes; lane 2, 0.5 minute; lane 3, 1 minute; lane 4, 2 minutes; lane 5, 3 minutes. 
     FIGS.  5 A- 5 B: Phosphorylation of enolase by thrombin-cleaved GstLck. 
     A. Phosphorylation of enolase as a function of recombinant p56 lck . Each reaction was carried out for 1 minute at 30° C., with 3 μg of enolase as substrate, and varying amounts of recombinant p56 lck . Lane 1 shows the result from 0 μg p56 lck ; lane 2, 0.01 μg p56 lck ; lane 3, 0.02 μg p56 lck ; lane 4, 0.03 μg p56 lck ; lane 5, 0.05 μg p56 lck ; lane 6, 0.07 μg p56 lck ; lane 7, 0.09 μg p56 lck ; lane 8, 0.11 μg p56 lck.    
     B. Time course of enolase phosphorylation by recombinant p56 lck . Each reaction was carried out at 30° C., with 0.01 μg of recombinant p56 lck  , and 3 μg of enolase as substrate. Lane 1 shows the result after 0 minutes; lane 2, 0.5 minutes; lane 3, 1 minute; lane 4, 2 minutes; lane 5, 3 minutes. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Experimental Procedures 
     Construction of p56 lck  expression vectors. A Stu-1 fragment from the mouse lck cDNA (Marth, J. D., Peet, R., Krebs, E. G., and Perlmutter, R. (1985)  CELL , 393-404) was cloned into the filled-in Eco-R1 site of the vector pGEX-2T (Pharmacia). The resulting plasmid pGEX-lck, is capable of expressing a glutathione-s-transferase/Lck (GstLck) fusion protein when transfected into  E. coli  cells. The GstLck coding sequence from pGEX-lck was amplified by PCR. The 5′ PCR primer 
     5′ TAT AAA TAT GTC CCC TAT ACT A 3′ 
     (SEQ. ID. NO.: 3), 
     was synthesized on an Applied Biosystems, Inc. model 380A synthesizer. This primer hybridizes to the 5′ region of the Gst coding sequence and encodes the ribosome binding site for the baculovirus polyhedrin gene. The 3′ PCR primer, 
     5′ CGT CAG TCA GTC ACG AT 3′ 
     (SEQ. ID. NO.: 4), 
     hybridizes to sequences immediately 3′ to the polylinker of pGEX-2T. This primer pair can be used to amplify any sequence cloned into the polylinker of pGEX-2T as a Gst/insert fusion. The amplified GstLck coding sequence was cloned into the vector pCR1000 (InVitrogen, Inc.) resulting in the plasmid pCR1000-GstLck. The pCR1000 vector was designed for easy cloning of PCR-amplified DNA, and was used as an intermediate cloning vector. A Not-1, BgI-II fragment from pCR1000-GstLck containing GstLck coding sequence was cloned into the Not-I, BgI-II sites of pVL1393. Lukow, V. A., and Summers, M. D. (1988) Virology 167, 56-71. The resulting plasmid, pVL1393-GstLck was used to produce a recombinant baculovirus in  Spodoptera frugiperda  9 (Sf9) cells following standard procedures. Summers, M. D., and Smith, G. E. (1987).  A Manual for baculovirus vectors and insect cell culture procedures , Texas A&amp;M bulletin No. 1555, (College Station, Tex. Agricultural Experimental Station and Texas A&amp;M University), 10-39. The cloning scheme used for the construction of pBMS-I is outlined in FIG.  1 A. The PCR primers used are the same described above. 
     Purification of GstLck from Sf9 cells. A 500 mL spinner culture of infected Sf9 cells in Excell-400 medium (JRH Biosciences) was harvested 48 hours after infection by centrifugation at 4° C. for 5 minutes. The cells were lysed in 50 mL of cold 50 mM Tris pH 8.0, 150 mM NaCI, 2 mM EDTA, 1 mM DTT, 1%(vol/vol) NP-40, 1 mM PMSF, 0.1 mg/mL aprotinin, 0.1 mg/mL leupeptin, 1 mM NaF, and 1 mM Na 3 VO 4  (lysis buffer). Insoluble material was removed by centrifugation at 10,000×g for 10 minutes at 4° C. The resulting cell lysate was determined to have a protein concentration of 9.5 mg/mL using the Coomassie Protein Assay Reagent (Pierce). 
     The GstLck protein was purified by a one-step affinity chromatograpy procedure using glutathione resin as described by the manufacturer (Pharmacia). For this experiment, 50 mg of Sf9 cellular lysate containing the GstLck protein was added to a 2-mL glutathione column and the unbound material removed by washing with 50 mL of lysis buffer. Bound proteins were eluted from the column with 2 column volumes of lysis buffer containing 5 mM glutathione. Eluted protein was diluted to 15 mL with lysis buffer and concentrated using a Centriprep 30 Concentrator unit (Amicon, Inc.). Two additional dilutions and concentrations were performed to remove the remaining glutathione. The concentrated protein was adjusted to 10% glycerol and stored at −70° C. This procedure yielded 28.0 mg of greater than 99% pure GstLck as determined by SDS-PAGE and Coomassie Blue staining analysis. 
     To obtain p56 lck  protein lacking the Gst peptide sequences, GstLck was digested with the proteolytic enzyme thrombin to generate cleaved p56 lck  (cp56 lck ). For this procedure 5 mg of thrombin was added to 20 mg of purified GstLck in a volume of 50 mL lysis buffer, containing 2.5 mM CaCl 2  for 1 hour at 25° C. To remove uncleaved GstLck and cleaved Gst, the products were mixed with 20 mL of glutathione resin. The glutathione resin was removed by centrifugation leaving the cp56 lck  in the supernatant. The yield from this procedure was approximately 5 mg of recombinant p56 lck  which was stored in 10% glycerol at − 70° C.    
     Immune-complex protein kinase assays. Analysis of protein kinase activity conducted on immune-complexes was carried out as previously described. Veillette, A., Horak, I. D., Horak, E. M., Bookman, M. A., and Bolen, J. A. (1988)  Mol. Cell. Biol.  8, 4353-4361. Briefly, immune-complexes formed from cellular lysates and the indicated antisera were collected by the addition of formalin-fixed  Staphyloccocus aureus  (Pansorbin, Calbiochem) and washed extensively in lysis buffer. Protein kinase reactions were initiated by the addition of 30 mL kinase buffer (20 mM MOPS pH 7, 5 mM MnCl 2 , 1 mM ATP) containing 12.5 μCi [γ- 32 P]-ATP (3000 Ci/mmol, New England Nuclear). The reactions were allowed to proceed for 5 minutes at room temperature and stopped by addition of an equal volume of 2 33  SDS loading buffer (0.125 M Tris-HCl pH 6.8, 4% (weight/vol) SDS, 20% (vol/vol) glycerol, 10% (vol/vol) 2-mercaptoethanol). The phosphorylated products in SDS loading buffer were heated for 5 minutes at 90° C. and analyzed by SDS-PAGE and autoradiography. The 32P-labeled bands of interest were excised from the gel and counted in a Beckman LS6000TA liquid scintillation counter. 
     Soluble protein kinase assays. The enzymatic activity of GstLck and cp56 lck  were evaluated by their capacity to phosphorylate the Lck exogenous substrate rabbit muscle enolase (Sigma). To determine the time course of enolase phosphorylation, 3 μg of GstLck or 1 μg of cp56 lck  was added to 100 μl of kinase buffer containing 12 μg enolase and 25 μgCi [γ −32 P]-ATP and the reactions were conducted at 30° C. for the indicated times. At each point, 10 μL of the reaction mix was removed, added to 30 μL of 2×SDS loading buffer and heated for 5 minutes at 90° C. The reaction products were analyzed by SDS-PAGE and autoradiography. The bands corresponding to enolase were excised from the gel and counted by liquid scintillation spectroscopy. To determine the K m  for enolase, serial dilutions of enolase were added to kinase buffer containing 5 μCi [γ −32 P]-ATP, and either 0.1 μg of GstLck or 0.01 μg of cp56 lck  were added per reaction. Reaction conditions and the counts incorporated into enolase were determined as described above. For the K m  determination of ATP, a 1:10 dilution of [γ −32 P]-ATP was added to kinase buffer containing 3 μg enolase. For each ATP dilution, 1 μg of cp56 lck  was added in a total volume of 30 μL and reacted for 30 seconds at 30° C. Reactions were stopped by addition of 30 μL of 2×SDS loading buffer and heated to 90° C. The reaction products were analyzed by SDS-PAGE, the phosphorylated proteins visualized by autoradiography, and  32 P incorporation determined by liquid scintillation spectroscopy of the excised bands. 
     Other biochemical assays and materials. Lck immunoblot analysis was conducted as previously described using rabbit anti-lck antisera. Veillette, A., Bookman, M. A., Horak, E. M., and Bolen, J. A. (1988) CELL  55, 301-308. Partial proteolytic peptide analysis using  Staphylococcus aureus  V8 protease (Pierce) has also been previously described. Veillette, A., Horak, I. D., Horak, E. M., Bookman, M. A., and Bolen, J. A. (1988)  Mol. Cell. Biol ., 4353-4361; Marth, J. D., Cooper, J. A., King, C. S., Ziegler, S. F., Tinker, D. A., Overell, R. A., Krebs, E. G., and Perlmutter, R. M. (1988)  Mol. Cell. Biol ., 540-550. The human T-cell lymphoma cell line CEM was grown in RPMl 1640 media supplemented with 10% (vol/vol) fetal bovine serum and antibiotics (penicillin/streptomycin). For immunoprecipitation experiments, the cells were washed in phosphate buffered saline, collected by centrifugation, lysed in lysis buffer, and adjusted to 1 mg/ml prior to addition of anti-Lck antisera. Antisera directed against Gst was prepared by immunization of rabbits with purified Gst. Antisera directed against Lck amino acids 39-58 has been previously described. Veillette, A., Bookman, M. A., Horak, E. M., and Bolen, J. A. (1988)  Cell  55, 301-308. 
     Results 
     Construction of expression vectors. FIG. 1A outlines the cloning strategy used to create the expression vector pBMS-I. The Gst coding sequence from pGEX-2T was cloned by PCR amplification, and ligated into the baculovirus expression vector pVL1393. The 5′ PCR primer was designed to optimize translation of the Gst coding sequence in Sf9 cells. This was accomplished by changing the sequence surrounding the initiation methionine of Gst to encode the ribosomal binding site of the baculovirus polyhedrin gene. The pBMS-I polylinker contains 9 unique cloning sites, and can be used to make a recombinant baculovirus that expresses inserts as a Gst fusion protein in Sf9 cells. 
     The fusion junction of the GstLck coding sequences cloned into pVL1393 is schematically shown in FIG.  1 B. The thrombin cleavage site is also indicated. This plasmid pVL1393-GstLck was used to make a recombinant baculovirus that expressed high levels of the GstLck fusion protein in Sf9 cells. Thrombin cleavage of GstLck protein resulted in a recombinant p56 lck  (cp56 lck ) molecule containing an additional 13 amino acids at the Lck amino-terminus. These additional amino acids had no apparent affect on the in vitro enzymatic activity of recombinant p56 lck . This was determined by comparing the immune-complex protein kinase activities of cp56 lck  with that of wild-type p56 lck  expressed in Sf9 cells. 
     Purification of GstLck from Sf9 cells. Total detergent lysates were made from Sf9 cells expressing the GstLck fusion protein as outlined in Experimental Procedures. Lysate containing GstLck was bound to a glutathione-sepharose column and eluted with 5 mM glutathione in lysis buffer. The glutathione-bound products from this column were analyzed by Coomassie staining following fractionation on SDS polyacrylamide gels. As shown in FIG. 2A, a single polypetide of approximately 83 kDa was observed which corresponds to the expected size for the GstLck fusion protein. Following thrombin cleavage (FIG. 2A, lane 3), the recombinant Lck protein was observed to migrate as two closely spaced bands at approximately 56 kDa. 
     Functional analysis of GstLck and cp56 lck . To evaluate the kinase activity of the purified GstLck and cp56 lck  proteins, protein kinase assays were performed. The results of these reactions (FIG. 2B) demonstrated that purified GstLck and cp56 lck  maintained their autophosphorylation capacity. As expected, no kinase activity was detected in purified preparations of Gst. The data shown in FIG. 2C represents the corresponding Lck immunoblot using polyclonal rabbit antibodies against the p56 lck  unique region. Based on the relative amounts of Lck protein detected in the kinase reactions, it appears that the specific activity of the cp56 lck  may be slightly higher than that of the GstLck fusion protein. Anti-phosphotyrosine immunoblot analysis of similar reaction products generated using non-radioactive ATP demonstrated that the autophosphorylation products (as well as the phosphorylation of exogenous protein substrate enolase used in other experiments) were phosphorylated on tyrosine residues. Additionally, partial V8 peptide analysis of the autophosphorylation products of the GstLck and cp56 lck  reactions yielded major V8 phosphopeptides indistinguishable from that of T-cell derived p56 lck  autophosphorylated in immune-complex kinase assays. 
     The level of GstLck enzymatic activity was also compared to that of wild type p56 lck  immunoprecipitated from T-cell detergent lysates. For these experiments, GstLck was precipitated from infected Sf9 detergent lysates with anti-Lck antisera, anti-Gst antisera, or with glutathione-Sepharose beads. The p56 lck  from T-cell lysates was immunoprecipitated with anti-Lck antisera. The various complexes were washed extensively with lysis buffer and divided into two equal aliquots. One aliquot was used to perform protein kinase assays (FIG. 3B) while the other aliquot was used for Lck immunoblot analysis (FIG.  3 A). The results of this experiment demonstrate that precipitation of the GstLck protein using either antibodies or glutathione beads yielded molecules with similar specific activities as assessed by autophosphorylation. Comparison with p56 lck  derived from T-cells showed that the specific activity of the Sf9 derived GstLck protein was significantly higher. 
     To further characterize the kinetic parameters of GstLck and cp56 lck , kinase activity of the fusion protein and cleaved enzyme was studied using rabbit muscle enolase as an exogenous substrate. As shown by the data presented in FIG. 4, the phosphorylation of enolase by GstLck was found to be both time and concentration dependent. Similar results were obtained for cp56 lck  (FIG.  5 ). The K m  and V max  values for ATP and enolase were determined using a reaction time of 30 seconds and the results summarized in Table 1. The affinity of cp56 lck  for enolase was found to be approximately 10-fold higher then that of GstLck. More critically the K m  and V max  values determined for cp56 lck  are comparable to values obtained for other src family members. 
     Attempts to produce functional GstLck in  E. coli  were unsuccessful. The resulting fusion protein was expressed, but it lacked detectable protein kinase activity and was found to be insoluble in detergents. The latter feature is common to expression of many eukaroytic proteins in bacteria. Marston, A. O. (1986)  J. Biochem , 240, 1-12; Miller, D. W., Saher, P., and Miller, L. K. (1986) in  Genetic Engineering , vol. 8, pp. 277-298, Plenum, N.Y.; Miller, L. K. (1989) in Ann. Rev. Microbiol. 42, 177-199. Among the advantages of expression of eukaryotic proteins in Sf9 cells is the capacity of these cells to allow protein folding and post-translational modification that maintain protein solubility. In the case of Lck, expression of the wild-type p56 lck  in Sf9 cells using conventional baculovirus expression vectors has shown that Lck is myristylated and phosphorylated on serine and threonine residues. Thomas, J. E., Soriano, P., and Brugge, J. S. (1991)  Science  254,.568-571. Since Lck in this system is expressed as a fusion protein with Gst at the aminoterminus, it is unlikely that myristylation occurs. We have not determined whether the GstLck is phosphorylated on serine or threonine residues. 
     Discussion 
     The lck coding sequences were ligated downstream from the Gst coding region in-frame to yield a plasmid capable of encoding a Gst-p56 lck  fusion protein. The p56 lck  produced in this manner was found to be a highly active protein kinase, and exhibited the expected biochemical properties of a member of the src family. 
     Analysis of both the GstLck fusion protein as well as the cp56 lck  indicated that each retained significant protein tyrosine kinase activity as measured by autophosphorylation and tyrosine phosphorylation of the exogenous substrate rabbit muscle enolase. Importantly, the Gst sequences, whether fused to Lck or following cleavage from the kinase with thrombin, were not phosphorylated in immune-complex kinase assays or in kinase assays conducted in solution. Both the GstLck and the cp56 lck  were found to have substantially higher specific activities than p56 lck  derived from T-cells when measured by immune-complex protein kinase assays. The altered specific activity is likely to be the result of diminished carboxy-terminal tyrosine (tyrosine 505) phosphorylation for Lck in Sf9 cells although we have not determined the phosphorylation sites of Lck in these cells. Veillette, A., Horak, I. D., Horak, E. M., Bookman, M. A., and Bolen, J. A. (1988)  Mol. Cell. Biol . 8, 4353-4361; Marth, J. D., Cooper, J. A., King, C. S., Ziegler, S. F., Tinker, D. A., Overell, R. A., Krebs, E. G., and Perlmutter, R. M. (1988)  Mol. Cell. Biol . 8, 540-550. The lack of tyrosine 505 phosphorylation of Lck like that observed with Sf9-derived pp60 c-src  (Morgan, D. O., Kaplan, J. M., Bishop, J. M., and Varmus, H. E. (1989) CELL  57, 775-786), is probably attributable to the absence of expression of other tyrosine protein kinases such as Csk that are thought to phosphorylate the Src class of kinases at this site. Okada, M., and Nakagawa, H. (1989)  J. Biol. Chem . 264, 20886-20893; Okada, M., and Nakagawa, H. (1988)  Biochem. Biophys. Res. Commun . 154, 796-7636 802. 
     From 50 mg of total 29 protein lysate, the foregoing procedure purified 280 mg of greater than 99% pure (by silver and Coomassie staining) recombinant p56 lck . From one liter of infected Sf9 cells, this system produced approximately 8-10 mg of purified recombinant Lck. 
     The foregoing procedures were also used to produce GstLynB, GstSyk, GstBlk, GstFyn, and GstYes fusion proteins with comparable results and yields to that reported here for Lck. These results are reported below in Tabel I. 
     The abbreviations used throughout this specification are defined as follows. 
     
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 ATP 
                 adenosine triphosphate 
               
               
                   
                 DNA 
                 deoxyribonucleic acid 
               
               
                   
                 DTT 
                 dithiothreitol 
               
               
                   
                 MOPS 
                 (3-[N-morpholino]propanesulfonic acid) 
               
               
                   
                 PCR 
                 polymerase chain reaction 
               
               
                   
                 PAGE 
                 polyacrylamide gel electrophoresis 
               
               
                   
                 PMSF 
                 phenylmethylsulfonyl fluoride 
               
               
                   
                 SDS 
                 sodium dodecyl sulfate 
               
               
                   
                   
               
             
          
         
       
     
     The gene for GST can be cleaved by enzymes at the positions (“Pos.”) shown in Table 2. Such nucleic acid fragments can be used to generate partial Gst polypeptides in the fusion proteins of the present invention. 
     
       
         
               
               
             
           
               
                   
               
             
             
               
                  11 
                 EcoN1 
               
               
                  13 
                 Bfa1 
               
               
                  13 
                 BsiY1 
               
               
                  13 
                 Bs11 
               
               
                  13 
                 Mae1 
               
               
                  13 
                 Rma1 
               
               
                  17 
                 BsmF 1 
               
               
                  26 
                 EcoR1* 
               
               
                  26 
                 Tsp509 1 
               
               
                  29 
                 Mse1 
               
               
                  33 
                 Asu1 
               
               
                  33 
                 BsiZ1 
               
               
                  33 
                 Cfr13I 
               
               
                  33 
                 Dra11 
               
               
                  33 
                 Eco01091 
               
               
                  33 
                 Nsp1V 
               
               
                  33 
                 Sau96I 
               
               
                  35 
                 BsuR1 
               
               
                  35 
                 Hae111 
               
               
                  35 
                 Pa11 
               
               
                  36 
                 Pss1 
               
               
                  51 
                 Taq1 
               
               
                  51 
                 TthHB81 
               
               
                  65 
                 Bcq1 
               
               
                  80 
                 Eam11041 
               
               
                  80 
                 Ear1 
               
               
                  80 
                 Ksp6321 
               
               
                  85 
                 Mbo11 
               
               
                  95 
                 Ms1 1 
               
               
                  97 
                 Mbo11 
               
               
                 102 
                 Hin61 
               
               
                 102 
                 HinP11 
               
               
                 102 
                 HinP1 
               
               
                 104 
                 Acc11 
               
               
                 104 
                 Bsh1236 1 
               
               
                 104 
                 Bsp501 
               
               
                 104 
                 BstU1 
               
               
                 104 
                 Cfo1 
               
               
                 104 
                 FnuD11 
               
               
                 104 
                 Hha1 
               
               
                 104 
                 Mvn1 
               
               
                 104 
                 Tha1 
               
               
                 121 
                 AciI 
               
               
                 124 
                 Hph1 
               
               
                 139 
                 EcoR1* 
               
               
                 139 
                 Tsp509 1 
               
               
                 154 
                 Mbo11 
               
               
                 188 
                 Mse1 
               
               
                 190 
                 EcoR1* 
               
               
                 190 
                 Tsp509 1 
               
               
                 193 
                 Hph1 
               
               
                 193 
                 Mse1 
               
               
                 205 
                 BsmA1 
               
               
                 206 
                 Cfr1 
               
               
                 206 
                 Eae1 
               
               
                 208 
                 Ba11 
               
               
                 208 
                 BsuR1 
               
               
                 208 
                 Hae111 
               
               
                 208 
                 Msc1 
               
               
                 208 
                 Pa11 
               
               
                 216 
                 Mae11 
               
               
                 226 
                 Alu1 
               
               
                 239 
                 Af1111 
               
               
                 243 
                 Nla111 
               
               
                 243 
                 Nsp75241 
               
               
                 243 
                 NspH1 
               
               
                 243 
                 Nsp1 
               
               
                 287 
                 Bsq1 
               
               
                 292 
                 BsrB 1 
               
               
                 319 
                 Taq1 
               
               
                 319 
                 TthHB81 
               
               
                 323 
                 EcoR1* 
               
               
                 323 
                 Tsp509 1 
               
               
                 333 
                 BsmA1 
               
               
                 367 
                 Dde1 
               
               
                 375 
                 Alu1 
               
               
                 394 
                 Asp7001 
               
               
                 394 
                 Xmn1 
               
               
                 398 
                 Asu11 
               
               
                 398 
                 Bpu141 
               
               
                 398 
                 BsiC1 
               
               
                 398 
                 Bsp1191 
               
               
                 398 
                 BstB1 
               
               
                 398 
                 Csp451 
               
               
                 398 
                 Lsp1 
               
               
                 398 
                 Nsp7524V 
               
               
                 398 
                 NspV 
               
               
                 398 
                 Sfu1 
               
               
                 398 
                 Taq1 
               
               
                 398 
                 TthHB81 
               
               
                 402 
                 BspA1 
               
               
                 402 
                 Dpn11 
               
               
                 402 
                 Kzo91 
               
               
                 402 
                 Mbo1 
               
               
                 402 
                 Nde11 
               
               
                 402 
                 Sau3A1 
               
               
                 404 
                 Dpn1 
               
               
                 412 
                 Mbo11 
               
               
                 427 
                 Mse1 
               
               
                 428 
                 Aha111 
               
               
                 428 
                 Dra1 
               
               
                 428 
                 SwaI 
               
               
                 434 
                 Fba1 
               
               
                 434 
                 Fok1 
               
               
                 435 
                 Bc11 
               
               
                 435 
                 BsiQ1 
               
               
                 435 
                 BspA1 
               
               
                 435 
                 Dpn11 
               
               
                 435 
                 Kzo91 
               
               
                 435 
                 Mbo1 
               
               
                 435 
                 Nde11 
               
               
                 435 
                 Sau3A1 
               
               
                 437 
                 Dpn1 
               
               
                 440 
                 Fba1 
               
               
                 441 
                 Mae111 
               
               
                 442 
                 Nla111 
               
               
                 445 
                 Hph1 
               
               
                 462 
                 Nla111 
               
               
                 478 
                 Hga1 
               
               
                 495 
                 Af11 
               
               
                 495 
                 Asu1 
               
               
                 495 
                 Ava11 
               
               
                 495 
                 Bme181 
               
               
                 495 
                 BsiZ1 
               
               
                 495 
                 Cfr13I 
               
               
                 495 
                 Eco47I 
               
               
                 495 
                 Eco471 
               
               
                 495 
                 Nla111 
               
               
                 495 
                 NspH11 
               
               
                 495 
                 Nsp1V 
               
               
                 495 
                 Sau96I 
               
               
                 495 
                 Sin1 
               
               
                 497 
                 BscB1 
               
               
                 497 
                 NlaIV 
               
               
                 501 
                 SfaN1 
               
               
                 506 
                 DsaV 
               
               
                 506 
                 EcoR11 
               
               
                 508 
                 Apy1 
               
               
                 508 
                 BsiL1 
               
               
                 508 
                 BstN1 
               
               
                 508 
                 BstO1 
               
               
                 508 
                 Mval 
               
               
                 508 
                 ScrF1 
               
               
                 523 
                 EcoR1* 
               
               
                 523 
                 Fok1 
               
               
                 523 
                 Tsp509 1 
               
               
                 536 
                 Mse1 
               
               
                 537 
                 Aha111 
               
               
                 537 
                 Dra1 
               
               
                 543 
                 Mae11 
               
               
                 553 
                 Alu1 
               
               
                 563 
                 EcoR1* 
               
               
                 563 
                 Tsp509 1 
               
               
                 573 
                 Csp61 
               
               
                 574 
                 Afa1 
               
               
                 574 
                 Rsa1 
               
               
                 574 
                 Sca1 
               
               
                 602 
                 Nla111 
               
               
                 603 
                 BsuR1 
               
               
                 603 
                 Hae111 
               
               
                 603 
                 Pa11 
               
               
                 610 
                 BsiY1 
               
               
                 610 
                 Bs11 
               
               
                 615 
                 BspW1 
               
               
                 615 
                 Mwo 1 
               
               
                 625 
                 Mae11 
               
               
                 629 
                 Fok1 
               
               
                 636 
                 AciI 
               
               
                 656 
                 Mn11 
               
               
                 657 
                 BspA1 
               
               
                 657 
                 BstY1 
               
               
                 657 
                 Dpn11 
               
               
                 657 
                 Kzo91 
               
               
                 657 
                 Mbo1 
               
               
                 657 
                 Mf11 
               
               
                 657 
                 Nde11 
               
               
                 657 
                 Sau3A1 
               
               
                 657 
                 Xho11 
               
               
                 659 
                 Dpn1 
               
               
                 665 
                 Alw1 
               
               
                 665 
                 BscB1 
               
               
                 665 
                 NlaIV 
               
               
                 667 
                 AciI 
               
               
                 668 
                 Alw1 
               
               
                 669 
                 Acc11 
               
               
                 669 
                 Bsh1236 1 
               
               
                 669 
                 Bsp501 
               
               
                 669 
                 BstU1 
               
               
                 669 
                 FnuD11 
               
               
                 669 
                 Mvn1 
               
               
                 669 
                 Tha1 
               
               
                 673 
                 BamH1 
               
               
                 673 
                 BspA1 
               
               
                 673 
                 BstY1 
               
               
                 673 
                 Dpn11 
               
               
                 673 
                 Kzo91 
               
               
                 673 
                 Mbo1 
               
               
                 673 
                 Mf11 
               
               
                 673 
                 Nde11 
               
               
                 673 
                 Sau3A1 
               
               
                 673 
                 Xho11 
               
               
                 675 
                 BscB1 
               
               
                 675 
                 Dpn1 
               
               
                 675 
                 NlaIV 
               
               
                 677 
                 BsaJ1 
               
               
                 677 
                 Bsa11 
               
               
                 677 
                 DsaV 
               
               
                 677 
                 Sec1 
               
               
                 678 
                 Aqu1 
               
               
                 678 
                 Ava1 
               
               
                 678 
                 Bco1 
               
               
                 678 
                 BsaJ1 
               
               
                 678 
                 Bas11 
               
               
                 678 
                 Cfr91 
               
               
                 678 
                 DsaV 
               
               
                 678 
                 Eco881 
               
               
                 678 
                 PspA1 
               
               
                 678 
                 Sec1 
               
               
                 678 
                 Xcy1 
               
               
                 678 
                 Xma1 
               
               
                 679 
                 Aha1 
               
               
                 679 
                 Bcn1 
               
               
                 679 
                 Hap11 
               
               
                 679 
                 Hpa11 
               
               
                 679 
                 Msp1 
               
               
                 679 
                 Nci1 
               
               
                 679 
                 ScrF1 
               
               
                 680 
                 Aha1 
               
               
                 680 
                 Bcn1 
               
               
                 680 
                 Nci1 
               
               
                 680 
                 ScrF1 
               
               
                 680 
                 Sma1 
               
               
                 681 
                 Alw1 
               
               
                 683 
                 Apo1 
               
               
                 683 
                 EcoR1* 
               
               
                 683 
                 EcoR1 
               
               
                 683 
                 Tsp509 1 
               
               
                   
               
             
          
         
       
     
     
       
         
           
             8 
           
           
             
               693 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             
               CDS 
                1..693 
             
             1
ATG TCC CCT ATA CTA GGT TAT TGG AAA ATT AAG GGC CTT GTG CAA CCC       48
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
  1               5                  10                  15
ACT CGA CTT CTT TTG GAA TAT CTT GAA GAA AAA TAT GAA GAG CAT TTG       96
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
             20                  25                  30
TAT GAG CGC GAT GAA GGT GAT AAA TGG CGA AAC AAA AAG TTT GAA TTG      144
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
         35                  40                  45
GGT TTG GAG TTT CCC AAT CTT CCT TAT TAT ATT GAT GGT GAT GTT AAA      192
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
     50                  55                  60
TTA ACA CAG TCT ATG GCC ATC ATA CGT TAT ATA GCT GAC AAG CAC AAC      240
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
 65                  70                  75                  80
ATG TTG GGT GGT TGT CCA AAA GAG CGT GCA GAG ATT TCA ATG CTT GAA      288
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
                 85                  90                  95
GGA GCG GTT TTG GAT ATT AGA TAC GGT GTT TCG AGA ATT GCA TAT AGT      336
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
            100                 105                 110
AAA GAC TTT GAA ACT CTC AAA GTT GAT TTT CTT AGC AAG CTA CCT GAA      384
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
        115                 120                 125
ATG CTG AAA ATG TTC GAA GAT CGT TTA TGT CAT AAA ACA TAT TTA AAT      432
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
    130                 135                 140
GGT GAT CAT GTA ACC CAT CCT GAC TTC ATG TTG TAT GAC GCT CTT GAT      480
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145                 150                 155                 160
GTT GTT TTA TAC ATG GAC CCA ATG TGC CTG GAT GCG TTC CCA AAA TTA      528
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
                165                 170                 175
GTT TGT TTT AAA AAA CGT ATT GAA GCT ATC CCA CAA ATT GAT AAG TAC      576
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
            180                 185                 190
TTG AAA TCC AGC AAG TAT ATA GCA TGG CCT TTG CAG GGC TGG CAA GCC      624
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
        195                 200                 205
ACG TTT GGT GGT GGC GAC CAT CCT CCA AAA TCG GAT CTG GTT CCG CGT      672
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg
    210                 215                 220
GGA TCC CCG GGA ATT CAT CGT                                          693
Gly Ser Pro Gly Ile His Arg
225                 230 
           
           
             
               231 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
             
               not provided 
             
             2
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
  1               5                  10                  15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
             20                  25                  30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
         35                  40                  45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
     50                  55                  60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
 65                  70                  75                  80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
                 85                  90                  95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
            100                 105                 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
        115                 120                 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
    130                 135                 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145                 150                 155                 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
                165                 170                 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
            180                 185                 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
        195                 200                 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg
    210                 215                 220
Gly Ser Pro Gly Ile His Arg
225                 230 
           
           
             
               22 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             3
TATAAATATG TCCCCTATAC TA                                              22 
           
           
             
               17 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             4
CGTCAGTCAG TCACGAT                                                    17 
           
           
             
               58 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             
               CDS 
                1..57 
             
             5
CTG GTT CCG CGT GGA TCC CGG GTA CCT TCT AGA ATT CCG GAG CGG CCG       48
Leu Val Pro Arg Gly Ser Arg Val Pro Ser Arg Ile Pro Glu Arg Pro
  1               5                  10                  15
CTG CAG ATC T                                                         58
Leu Gln Ile 
           
           
             
               19 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
             
               not provided 
             
             6
Leu Val Pro Arg Gly Ser Arg Val Pro Ser Arg Ile Pro Glu Arg Pro
  1               5                  10                  15
Leu Gln Ile 
           
           
             
               57 base pairs 
               nucleic acid 
               single 
               linear 
             
             
               cDNA 
             
             
               not provided 
             
             
               CDS 
                1..57 
             
             7
CTG GTT CCG CGT GGA TCC CCG GGA ATT CCT CTC TAC ATT CCT TCA GGG       48
Leu Val Pro Arg Gly Ser Pro Gly Ile Pro Leu Tyr Ile Pro Ser Gly
  1               5                  10                  15
ATC ATG GGC                                                           57
Ile Met Gly 
           
           
             
               19 amino acids 
               amino acid 
               linear 
             
             
               protein 
             
             
               not provided 
             
             8
Leu Val Pro Arg Gly Ser Pro Gly Ile Pro Leu Tyr Ile Pro Ser Gly
  1               5                  10                  15
Ile Met Gly