WO1993019091A1 - Proteines de fusion tripartites de glutathione s-transferase - Google Patents

Proteines de fusion tripartites de glutathione s-transferase Download PDF

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WO1993019091A1
WO1993019091A1 PCT/AU1993/000105 AU9300105W WO9319091A1 WO 1993019091 A1 WO1993019091 A1 WO 1993019091A1 AU 9300105 W AU9300105 W AU 9300105W WO 9319091 A1 WO9319091 A1 WO 9319091A1
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protein
binding
fusion protein
gst
amino acid
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PCT/AU1993/000105
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Donald Bruce Smith
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Amrad Corporation Limited
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C12N9/1088—Glutathione transferase (2.5.1.18)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/705—Fusion polypeptide containing domain for protein-protein interaction containing a protein-A fusion

Definitions

  • This invention relates to the expression in bacteria such as Escherichia coli of foreign polypeptides as fusions with glutathione-S-transferase (GST).
  • GST glutathione-S-transferase
  • a fusion protein having a foreign polypeptide component fused to the enzyme glutathione-S- transferase (E.C. 2.5.1.18), preferably to the carboxy-terminal of the enzyme avoids several of the difficulties associated with known fusion proteins, for instance fusions wherein the foreign polypeptide is expressed as a fusion with E.coli ⁇ -galactosidase, in that the GST fusion proteins are generally soluble and can be purified from bacterial lysates under non-denaturing conditions, for example by affinity chromatography on a column of immobilised glutathione.
  • the GST enzyme in the fusion protein may be derived from the parasite helminth Schistosoma japonicum, or it may be derived from other species including humans and other mammals.
  • the GST fusion proteins disclosed in International Patent Application No. PCT/AU88/00164 may be used as such, since the foreign polypeptide component thereof often retains its antigenicity and functional activity.
  • the fusion protein may be cleaved to provide the foreign polypeptide as a synthesis product, and when the production of such a synthetic polypeptide is desired a cleavable link may be provided in the fusion protein between the glutathione-S- transferase component and the foreign polypeptide component.
  • the cleavable link is preferably one which can be cleaved by a site-specific protease such as thrombin, blood coagulation Factor Xa, or the like.
  • this earlier application discloses a fusion protein comprising a first amino acid sequence corresponding to the enzyme glutathione- S-transferase and, preferably fused to the COOH-terminus thereof, a second amino acid sequence corresponding to a foreign polypeptide (and optionally a cleavable link between these two components), as well as recombinant DNA molecules, expression vectors, and host cells for use in the production of such a fusion protein.
  • this earlier application discloses an expression vector (such as a bacterial plasmid) for use in the production of a foreign polypeptide, wherein the vector has inserted therein a nucleotide sequence capable of being expressed as the glutathione-S-transferase enzyme followed by at least one restriction endonuclease recognition site for insertion of a nucleotide sequence capable of being expressed as a foreign polypeptide fused with the COOH- terminus of the glutathione-S-transferase enzyme, optionally with a sequence capable of being expressed as a cleavable link between the enzyme and the foreign polypeptide.
  • a nucleotide sequence capable of being expressed as the glutathione-S-transferase enzyme followed by at least one restriction endonuclease recognition site for insertion of a nucleotide sequence capable of being expressed as a foreign polypeptide fused with the COOH- terminus of the glutathione-S-transferase enzyme, optional
  • expression of the fusion proteins by the expression vectors is under die control of the tac promoter which enables inducible, high-level production of these fusion proteins.
  • the expression vectors contain the lac Iq gene, so that they can be used in any E.coli strain.
  • polypeptides expressed in E.colias fusions with GST 1 have proven useful for the analysis of protein-DNA and protein-protein interactions. Part of the reason for this is that, in contrast to many other expression systems, the purification of GST fusion proteins involves non-denaturing conditions so that the expressed polypeptide is recovered in a relatively native state and retains at least some of its normal properties. Examples include GST fusions with GCN4 2 and PEA3 3 that behave at site-specific DNA binding proteins with properties similar to those of their normal counterparts, while fusions with the retinoblastoma gene product (Rb) 4,5 , Adenovirus E1A 6 , c-Kit 7 or the TATA binding protein (TBP) 8 retain specific interactions with other proteins.
  • Rb retinoblastoma gene product
  • TBP TATA binding protein
  • GST fusion proteins have been used to identify and characterise previously unknown properties of polypeptides.
  • nucleotide sequences that are recognised by myogenin homomers 9 , MyoD 10 and c-Myc 11 have been delineated through Polymerase Chain Reaction (PCR) amplification of DNA selected by GST-fusion proteins from mixtures containing random sequences, while fusions with TBP 12 , Rb 4 ' 13 and c- Myc 6 have been used to characterise previously unknown interactions with other proteins.
  • the GST expression vector PGEX-2T 1 has been modified so that it directs the expression of glutathione-S-transferase (GST) fusion proteins that also contain one or more IgG binding domains from Staphylococcus aureus protein A.
  • GST glutathione-S-transferase
  • Such tripartite fusion proteins can be detected directly with commercially available antibody-enzyme conjugates, thus simplifying the use of GST fusion proteins as probes for the analysis of protein-protein interactions.
  • GST COOH-terminus is followed by one or more of the five naturally occurring IgG binding domains from Staphylococcus aureus protein A.
  • the present invention provides a fusion protein comprising a first amino acid sequence corresponding to a glutathione-S-transferase enzyme fused at its COOH terminus with a second amino acid sequence corresponding to a binding entity, followed by a third amino acid sequence corresponding to a different polypeptide fused with said binding entity.
  • a fusion protein comprising a first amino acid sequence corresponding to glutathione-S-transferase enzyme fused at its COOH terminus with a second amino acid sequence corresponding to at least one immunoglobulin binding entity such as an immunoglobulin binding protein or one or more binding domains thereof, followed by a third amino acid sequence corresponding to a different polypeptide fused with said immunoglobulin binding entity.
  • the present invention also provides a recombinant DNA molecule comprising a nucleotide sequence which codes on expression for a fusion protein in which a binding entity is fused with the COOH-terminus of a glutathione-S-transferase enzyme, followed by a different polypeptide fused with said binding entity.
  • a preferred embodiment of this aspect of the present invention provides a recombinant DNA molecule comprising a nucleotide sequence which codes on expression for a fusion protein in which at least one immunoglobulin binding entity such as an immunoglobulin binding protein or one or more binding domains thereof is fused with the COOH-terminus of a glutathione-S-transferase enzyme, followed by a different polypeptide fused with said immunoglobulin binding entity.
  • die fusion protein may also include a cleavable link, for example a link which can be cleaved by a site specific protease such as thrombin or blood coagulation Factor Xa.
  • a cleavable link is preferably included between the GST moiety and the binding entity; however, a cleavable link may additionally or alternatively be included between the binding entity and the polypeptide fused dierewith.
  • the present invention also provides expression vectors and host cells having inserted therein a recombinant DNA molecule in accordance with this invention, as well as methods of producing the fusion protein of this invention using such expression vectors and host cells as disclosed in detail in prior International Patent Application No. PCT/AU88/00164, the disclosure of which is incorporated by reference.
  • the present invention also provides an expression vector having inserted therein a nucleotide sequence capable of being expressed as a glutadiione-S-transferase enzyme followed by a binding entity fused with the COOH-terminus of said glutathione-S-transferase, and at least one restriction endonuclease recognition site for insertion of a further nucleotide sequence capable of being expressed as a different polypeptide fused with said binding entity.
  • Such an expression vector may, if desired, also include a nucleotide sequence capable of being expressed as a cleavable link as discussed above.
  • the present invention also provides an expression vector having inserted therein a nucleotide sequence capable of being expressed as a glutathione-S-transferase enzyme followed by at least one immunoglobulin binding entity such as an immunoglobulin binding protein or one or more binding domains thereof fused with the COOH-terminus of said glutathione-S-transferase, and at least one restriction endonuclease recognition site for insertion of a further nucleotide sequence capable of being expressed as a different polypeptide fused with said immunoglobulin binding entity.
  • this expression vector may also include a nucleotide sequence capable of being expressed as a cleavable link as discussed above.
  • the GST enzyme in the fusion protein may be derived from Schistosoma japonicum , or it may be derived from other species including humans and other mammals.
  • the binding entity which is included within the fusion protein in accordance witfi the present invention may be any member of a specific binding pair, including for example an antigen/specific antibody binding pair (especially an antigen/specific monoclonal antibody binding pair), and avidin/biotin binding pair, or preferably an immunoglobulin binding protein /immunoglobulin binding pair.
  • an antigen/specific antibody binding pair especially an antigen/specific monoclonal antibody binding pair
  • avidin/biotin binding pair or preferably an immunoglobulin binding protein /immunoglobulin binding pair.
  • the immunoglobulin binding entity included in preferred fusion proteins in accordance with this invention may be any protein having immunoglobulin binding affinity, more particularly binding affinity for immunoglobulin G (IgG).
  • immunoglobulin G immunoglobulin G
  • one or more immunoglobulin binding domains of such a protein may be incorporated in die fusion protein.
  • a particularly preferred binding entity comprises Staphylococcus aureus protein A or one or more of the IgG binding domains thereof, however the invention also encompasses the use of other immunoglobulin binding proteins such as Protein G, Protein GG and chimaeric Protein A/G. It will be appreciated that the immunoglobulin binding protein or binding domain(s) may be selected so as to optimise the binding to IgG in the intended application.
  • Protein G has a different spectrum of binding (as described by Lew et.al ⁇ 6 ), and the immunoglobulin binding entity may even be a synthetic IgG binding domain as described by Lowenalder et.al ⁇ 9 .
  • die present invention extends to such fusion proteins which incorporate any polypeptide or protein of interest as the different or “foreign” polypeptide.
  • this polypeptide or protein of interest may be a particular antigen, with the resulting fusion protein being useful in a diagnostic test method and kit for detection of specific antibody to the particular antigen in a sample, such as a serum sample, using the well-known enzyme immunoassay (EIA) techniques.
  • EIA enzyme immunoassay
  • the polypeptide or protein of interest may be a "reporter" entity, particularly an enzyme such as alkaline phosphatase, urease, horseradish peroxidase, or any other enzyme used in colorimetric or chemiluminescent determinations.
  • a fusion protein for example a GST/ Protein A/horseradish peroxidase fusion protein, can be used directly in various immunoassay procedures to detect antigen-antibody reactions by binding to the protein A moiety, and then directly detected by standard colorimetric or chemiluminescent methods. In this way, the requirement for various antibody/ reporter molecule conjugates (such as goat anti-mouse/HRPO or rabbit anti-human/HRPO conjugates) for use in such immunoassay procedures can be avoided.
  • the inclusion of the GST moiety in the fusion protein of the present invention enables the ready purification of the fusion protein by affinity chromatography on immobilised glutathione.
  • the fusion protein may be immobilised on a solid substrate by first coating the solid substrate with anti-GST antibody and then contacting the coated substrate with d e fusion protein.
  • This Example illustrates the construction of altered pGEX2T expression vectors that direct die expression of GST fusions containing one, two or three protein A IgG binding domains. These fusion proteins bind to IgG and can be detected using commercially available antibody-enzyme conjugates. Applications of these vectors to die analysis of protein-protein interactions are illustrated by experiments using an NH 2 -terminal region of SV40 T antigen as a probe on Western blots of cell extracts, and by binding assays of immobilised GST fusion proteins probed witii GST-protein A fusions.
  • Figure 1 shows detection of GST-protein A fusion proteins on
  • FIG. 2 shows the structure of pAGEX2T.
  • IPTG-inducible trp-lac hybrid promoter Ptac
  • GST mitochondrial
  • Th thrombin cleavage site
  • two copies of the S.aureus protein A IgG binding domain B(PA) followed by unique BamHI, Smal and EcoRI restriction sites are also represented.
  • the position of unique PstI and EcoRV restriction sites the gene encoding ⁇ - lactamase (amp r ), an origin of replication (ori), and an over-expressed allele of die lac repressor (lacl q ).
  • the nucleotide sequence of a single IgG binding domain and die polypeptide that it encodes are also given.
  • Figure 3 shows the sensitivity of detection of GST-protein A fusion proteins. Equal quantities of purified GST or GST-protein A (GST-PA) were applied to nitrocellulose from left to right as 5 ⁇ l drops in four-fold dilutions from 1 ⁇ g to 1 ng.
  • GST-PA GST-protein A
  • Air dried strips were probed with G53, a monoclonal antibody directed against GST followed by AP-conjugated goat anti-mouse antibodies (Anti-GST), with AP-conjugated rabbit antibodies (AP-Conjugated Rabbit Ab), witii eitiier R819, a rabbit antisera raised against Rb (Anti-Rb) or a non-specific rabbit serum (Sigma) (Rabbit Serum) followed by AP-conjugated goat anti-rabbit antibodies, or with Ab419, a monoclonal antibody specific for SV40 T antigen followed by AP-conjugated goat anti-mouse antibodies (Anti-TAg). After washing, enzyme activity was detected in sit .
  • Figure 4 shows the use of GST-protein A-SV40 TAg as a probe for protein-protein interactions on Western blots.
  • a Western blot of a HeLa whole cell extract was probed with GST-protein A(a) or GST-protein A-SV40 TAg (1- 272) (b) at a concentration of 25 ⁇ g/ml in NT buffer containing 150 mM, 500 mM or 1 M NaCl. After washing in the same buffer containing 150 mM NaCl, bound protein was detected by probing with AP-conjugated rabbit antibodies followed by enzyme detection in situ .
  • FIG. 5 shows that-Rb is amongst the proteins detected by GST- protein A-SV40 TAg.
  • Figure 6 shows the binding of protein A-TEF-1 to GST-SV40 TAg immobilised on beads.
  • Purified GST-Protein A-TEF1 (1-168) was cleaved witii thrombin and 1,3 or 10 ⁇ l incubated with glutathione-agarose beads carrying GST or GST-TAg (1-260). After washing, probe retained on the beads was analysed by probing Western blots with AP-conjugated rabbit antibodies and detection of enzyme activity in situ . Total represents 1.5 ⁇ l of probe. The triangles indicate die increasing quantity of probe from left to right.
  • Figure 7 shows the binding of complex probes to GST fusion proteins on beads.
  • a single IgG binding domain (domain B) of S.aureus protein A 20 was isolated by PCR amplification from the plasmid pRIT 5 (Pharmacia 21 ) using the oligonucleotides 5'-TGAGATCTGCGGATAACAAATTCAAC-3' and 5'- ACGGATCCTTTTGGTGCTTGAGCATC-3'.
  • Amplification was for 20 cycles of 94 °C 1 minute, 55 °C for 1% minutes and 74 °C for 1 % minutes using 25 ng pRIT5 and 100 ng each oligonucleotide in a solution containing 10 mM Tris (HC1) pH 8.3, 1.5 mM Mgcl 2 , 50 mM KCl, 200 ⁇ m dATP, dCTP, dGTP and dTTP and 2.5 units Thermus aquaticus DNA polymerase (Cetus).
  • the PCR product was incubated for 1 hour at 37 °C with the restriction endonuclease Sau3A in 20 mM Tris-HCl (pH 7.4), 5 mM MgCl 2 , 50 mM KCl and after electrophoresis through a 2% low-gelling-temperature agarose gel a 185 bp fragment was purified and ligated with BamHl cleaved and calf intestinal alkaline phosphatase treated pGEX-2T in a 10 ⁇ l reaction containing 100 ng of vector, 50 ng purified fragment, 20 mM Tris-HCl (pH 7.6), 5 mM MgCl 2 , 5 mM didiiothreitol, 1 mM ATP and 1 unit T4 DNA ligase (Pharmacia).
  • the ligation reaction was transformed into competent E.coli cells of the strain HB101 and spread onto agar plates containing 50 ⁇ g mM ampicillin. Individual colonies appearing after 15 hours growth at 37 °C were screened for tiieir ability to direct expression of an enlarged glutathione-S-transferase by inoculation into 1 ml of L-broth, growth for 4 hours with agitation at 37 °C, and a further 2 hours growth after the addition of isopropylthio-B-D-galactoside (IPTG) to 0.1 mM.
  • IPTG isopropylthio-B-D-galactoside
  • Cells were pelleted out of a 50 ⁇ l sample, heated at 90 °C for 2 minutes in SDS sample buffer and separated by electrophoresis through a 10% SDS - polyacrylamide gel. Proteins were visualised by staining with Coomassie Brilliant Blue and a clone identified that directs the expression of a 33 kDa polypeptide.
  • pGEX2T-TEFl (1-426) was constructed by isolating a 1.3 kb NcoI-BgUI fragment from pXJ40-TEFlA 23 , treating with the Klenow fragment of E.coli DNA polymerase I (Klenow) to fill in the ends, followed by litigation with Smal cleaved pGEX2T.
  • Klenow E.coli DNA polymerase I
  • the single PflMI restriction site in pGEX2T was removed by cleavage and religation after incubation with Klenow.
  • a BamHI- EcoRI fragment from pAGEX2T-SV40-TAg (1-272) was introduced into this vector, and using the unique PflMI restriction site within the SV40 coding sequences, die PflMI-EcoRI fragment was replaced with a PflMI-Hindlll fragment of PKT260 24 to produce the plasmid pGEX2T-SV40 TAg (1-260). Standard procedures were followed for manipulations of DNA and E.c ⁇ li transformation. GST fusion proteins were purified from bacterial cultures as described 1 .
  • Protein samples were separated by electrophoresis through 10% SDS- polyacrylamide gels and transferred to nitrocellulose at 4 °C in a buffer containing 25 mM Tris-HCl (pH 8.3), 192 mM glycine and 20% v/v methanol. Transfer was at 100 V for 1 hour or at 30 V for 15 hours, after which blots were blocked for 30 minutes at 20 °C in PBS (140 mM NaCl, 3 mM KCl, 8 mM Na 2 HPO 4 and 2 mM KH 2 PO 4 ) containing 5% non-fat dried milk powder.
  • PBS 140 mM NaCl, 3 mM KCl, 8 mM Na 2 HPO 4 and 2 mM KH 2 PO 4
  • blots were probed for 1 hour at 20 °C with a 1:5,000 dilution in PBS of an alkaline- phosphatase (AP) conjugated rabbit anti-chicken antibody (Chemicon). Blots were then washed three times in PBS with enzyme activity detected by incubation in die dark at 20 °C in a solution containing 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl 2 , 0.33 mg/ml nitro blue tetrazolium (Sigma) and 0.17 mg/ml 5-bromo-4-chloro-3-indolyl phosphate (Sigma).
  • AP alkaline- phosphatase conjugated rabbit anti-chicken antibody
  • fusion proteins The sensitivity of detection of fusion proteins was assessed by dot blots in which equal quantities of purified GST or GST-protein A were diluted in 150 mM NaCl, 50 mM Tris-HCl (pH 7.5) (NT buffer) containing 1% bovine serum albumin (fraction V, Sigma) (BSA) and applied in four-fold dilutions as 5 ⁇ l drops to nitrocellulose strips. After air drying, blots were blocked as above and probed for 1 hour at 20 °C with antisera diluted in NT buffer containing 3% BSA.
  • BSA bovine serum albumin
  • Blots were then rinsed three times in NT buffer, probed for 1 hour at 20 °C in NT buffer containing 3% BSA and a 1:5,000 dilution of AP-conjugated goat anti- mouse or anti-rabbit antibodies (Promega), and developed as above.
  • Cell pellets or aliquots of a HeLa whole cell extract 25 were heated to 90 ⁇ C in sample buffer, separated through 10% SDS-polyacryl amide gels and transferred to nitrocellulose or Immobilon-P (Millipore). After blocking as above, blots were cut into strips and probed for 2 hours at 20 °C with purified GST-protein A or GST-protein A-SV40 TAg (1-272) at a concentration of 25 ⁇ g/ml in NT buffer containing 3% BSA. Blots were washed three times in NT buffer and bound proteins then detected by probing with AP-conjugated rabbit antibodies, or with a pool of monoclonal antibodies raised against T antigen followed by AP- conjugated goat anti-mouse antibodies.
  • Thrombin cleavage of fusions was carried out while proteins were immobilised on beads (200 ⁇ l, containing up to 200 ⁇ g fusion protein) in 200 ⁇ l buffer containing 50 mM Tris-HCl (pH 8.0), 250 mM NaCl, 50 mM NaCitrate, 1 mM CaCl 2 , 1% BSA and 2 ⁇ g human thrombin (Sigma). After incubation at 20 °C for 1 hour beads were pelleted and the supernatant removed. The beads were washed once with 50 mM Tris-HCl (pH 8.0) and the combined supernatants stored at -80 °C.
  • a single IgG binding domain of S.aureus protein A (domain B) 20 was isolated by PCR amplification of DNA of the plasmid pRIT5 21 using oligonucleotide primers specific for domain B and inserted into the BamHI site of pGEX2TA The primers were designed such that the BamHI site was re-formed at a position corresponding to d e COOH-terminus of the IgG binding domain so tiiat further domains could be introduced in the same manner. E.coli transformants were identified containing plasmids that direct the expression of GST fusion proteins containing one, two or three binding domains.
  • This vector contains unique BamHI, Smal and EcoRI restriction sites at a position corresponding to the COOH-terminus of the second IgG binding domain and in d e same reading frame as in the parent plasmid pGEX2T, followed by termination codons in all three frames ( Figure 2). Fusions expressed using this vector consist of GST followed by a thrombin cleavage site, two protein A IgG binding sites and finally the protein of interest.
  • Enhanced sensitivities of less than 1 ng were observed when GST-protein A was probed first with an unconjugated antibody (Rabbit Serum, rabbit Anti-Rb or MAb419, an Anti-TAg monoclonal antibody) followed by the appropriate enzyme-conjugated second antibody. Amplification of signal in these cases may reflect polyvalent binding of enzyme-conjugated secondary antibodies to primary antibodies bound to the protein A domains. Similar sensitivities were observed for tripartite fusion proteins containing GST, protein A and portions of SV40 T antigen or of the transcriptional activator TEF- 1. This suggests that the presence of extraneous polypeptide sequences on both flanks of the protein A domains does not hinder the binding of antibodies.
  • pAGEX2T vector can be illustrated using an NH 2 -terminal region of SV40 T antigen (amino acids 1-272) that is known to interact with several different proteins 26,27,28 and when expressed as a GST fusion protein is able to autoregulate and transactivate the SV40 early and late promoters in in vitro transcription reactions 22 .
  • a DNA fragment encoding this region was inserted into pAGEX2T resulting in the expression of a tripartite fusion protein of 67 kDa.
  • this purified fusion protein was used as a probe on Western blots of a transcriptionally active HeLa whole cell extract 25 distinct bands over a range of molecular weights were recognised ( Figure 4, 0.15 M, b).
  • Blots incubated with the same probe in solutions containing higher concentrations of NaCl showed different patterns (0.5 M, 1.0 M) while only faint signals were observed on blots probed under the same conditions with GST protein A alone (a).
  • the strength of signal on these blots was reduced with lower concentrations of probe while the pattern obtained was influenced by the type of transfer membrane, the transfer conditions, post-transfer renaturation of proteins 29 and the type of protein blocker and solute concentrations used during probing (data not shown).
  • Nitrocellulose and Immobilon-P have different binding properties and it is presumed that a species present in both cell types interacts with the GST-protein A-SV40 T antigen fusion protein but transfers efficiently only to Immobilon-P where it obscures the interaction witii Rb.
  • a probe consisting of the protein A domains fused to the NH 2 -terminal 168 amino acids of TEF-1 does not bind to beads bearing GST alone but is retained on beads carrying GST fused to the first 260 amino acids of SV40 T antigen ( Figure 6).
  • This interaction is specific since a breakdown fragment of the protein A-TEF-1 fusion does not bind to either type of beads, and the intact fusion fails to bind to beads bearing GST fused to NH 2 -terminal or COOH- terminal fragments of TEF-1 or to entire TBP (data not shown).
  • both fusions possess DNA binding activities, the interaction does not appear to be mediated by DNA since the binding of probe to beads is not affected by the addition of DNA to 30 ⁇ g/ml or of DNAase I to 10 ⁇ g/ml.
  • Another illustration of this method takes advantage of cases where two or more cleaved GST-protein A fusion proteins are of different sizes and so can be used together in binding assays.
  • cleaved fusion proteins containing protein A fused to TBP or the NH 2 -terminal 272 amino acids of SV40 T antigen were mixed and used as a probe, binding to GST-SV40 TAg (1-260) beads above the background seen on GST beads was only observed for protein A-TBP ( Figure 7).
  • the GST expression vector pGEX2T has been modified in order to simplify the use of GST fusion proteins in the analysis of protein-protein interactions by producing tripartite fusion proteins that contain IgG binding domains.
  • Polypeptides expressed in this system can be purified on glutathione- agarose under non-denaturing conditions and can be detected without further modification using standard reagents and without the use of radioactive materials. This system is less disruptive and more general than some other methods.
  • Previous studies have used GST fusions as probes for protein-protein interaction after labelling by iodination 15,31 or biotinylation 14 . Such treatments might sometimes obscure interactions because of the incorporation of labelling moieties at binding sites.
  • An alternative non-disruptive method for detecting GST fusion proteins would be to use monoclonal antibodies specific for GST. Although this approach has been used to probe blots with GST-SV40 TAg (1-272) and obtain results similar to those obtained with a GST-protein A fusion, the method requires an additional specific reagent and extra manipulations. Such a detection system would also not allow the detection of cleaved GST fusion proteins as used in the binding experiments described above.
  • GST-protein A fusion proteins require either an en-zyme- linked antibody that itself binds to protein A, or else antibody that binds to protein A and an appropriate enzyme-linked second antibody.
  • GST-protein A fusions have been detected directly with AP-conjugated rabbit antibodies and indirectly using mouse monoclonal antibodies or rabbit serum followed by enzyme-linked goat anti-mouse or anti-rabbit antibodies. Fusion proteins could also be detected using antibodies from other species such as guinea-pig, cow, human, pig or horse, or might utilise other enzyme-conjugates or include biotin- avidin binding steps so as to further amplify the signal. Antibodies from species such as the goat, sheep, rat and chicken bind poorly to protein A 32 and so could not be used by themselves.
  • the binding properties of polypeptides may be affected by the presence of GST and IgG binding domains in the fusion proteins.
  • Removal of GST from a tripartite fusion protein containing amino acids 1-272 of SV40 T antigen by cleavage with thrombin did not affect the pattern of bands detected on a Western blot of HeLa cell proteins.
  • similar patterns were obtained when the same region expressed as a GST fusion protein, but lacking the protein A domains, was used as a probe and detected using a pool of monoclonal antibodies directed against GST.
  • GST fusion proteins have been observed to mirror the properties of their native counterparts in many instances 2'16 ' 22,31 .
  • T antigen might interact with TEF-1
  • TBP Herpes Simplex virus VP16 34
  • cytomegalovirus IE2 12 The possibility that SV40 T antigen might interact with TEF-1 was previously suggested by experiments indicating that the ability of T antigen to activate SV40 transcription is at least partially dependent on DNA sequences in the SV40 enhancer to which TEF-1 binds 22,33 .
  • Interactions between TBP and other virus-specified trans activators have already been demonstrated for Adenovirus EIA 8 , Herpes Simplex virus VP16 34 and cytomegalovirus IE2 12 .

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Abstract

Une protéine de fusion tripartite comprend une première séquence d'acides aminés correspondant à une enzyme glutathione-S-tranférase, placée à sa terminaison COOH, une deuxième séquence d'acides aminés correspondant à une entité de liaison, présentant notamment des caractéristiques immunologiques, et une troisième séquence d'acides aminés correspondant à un polypeptide différent fusionné avec l'entité de liaison. La protéine peut comporter un lien clivable placé entre la première et la deuxième et/ou la deuxième et la troisième séquences d'acides aminés. On décrit aussi des molécules d'ADN recombiné et des vecteurs d'expression codant pour ces protéines de fusion.
PCT/AU1993/000105 1992-03-18 1993-03-17 Proteines de fusion tripartites de glutathione s-transferase WO1993019091A1 (fr)

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WO1995006125A1 (fr) * 1993-08-23 1995-03-02 Applied Immune Sciences, Inc. RECEPTEUR CHIMERIQUE CONTENANT UN DOMAINE DE FIXATION DE L'IgG COMMUN AUX PROTEINES A ET G
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WO2000020606A1 (fr) * 1998-10-02 2000-04-13 Reimann Hansjoerg Procede de production de (poly)peptides a l'aide de variantes tronquees de l'antigene t de grande taille avec une terminaison t intacte
US6372249B1 (en) 1991-12-16 2002-04-16 Baylor College Of Medicine Senscent cell-derived inhibitors of DNA synthesis
WO2016186575A1 (fr) * 2015-05-15 2016-11-24 Agency For Science, Technology And Research Technologie de purification de protéine native
AT517379A1 (de) * 2015-06-26 2017-01-15 Technische Universität Graz Fusionsprotein
WO2019107896A1 (fr) * 2017-11-28 2019-06-06 울산과학기술원 Protéine de fusion comprenant une glutathion-s-transférase et une protéine ayant une affinité de liaison pour une cellule cible ou une protéine cible, et utilisation correspondante

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GENE, Volume 67, issued March 1988, (Elsevier Scientific Publishing Company, Netherlands), D. SMITH AND S. JOHNSON, "Single-Step Purification of Polypeptides Expressed in E. Coli as Fusions with Glutathione S-Transferase", see pages 31-40. *
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5596079A (en) * 1991-12-16 1997-01-21 Smith; James R. Mimetics of senescent cell derived inhibitors of DNA synthesis
US6372249B1 (en) 1991-12-16 2002-04-16 Baylor College Of Medicine Senscent cell-derived inhibitors of DNA synthesis
US6818215B2 (en) 1991-12-16 2004-11-16 James R. Smith Antibodies to senescent cell-derived inhibiters of DNA synthesis
WO1995006125A1 (fr) * 1993-08-23 1995-03-02 Applied Immune Sciences, Inc. RECEPTEUR CHIMERIQUE CONTENANT UN DOMAINE DE FIXATION DE L'IgG COMMUN AUX PROTEINES A ET G
WO2000020606A1 (fr) * 1998-10-02 2000-04-13 Reimann Hansjoerg Procede de production de (poly)peptides a l'aide de variantes tronquees de l'antigene t de grande taille avec une terminaison t intacte
WO2016186575A1 (fr) * 2015-05-15 2016-11-24 Agency For Science, Technology And Research Technologie de purification de protéine native
AT517379A1 (de) * 2015-06-26 2017-01-15 Technische Universität Graz Fusionsprotein
AT517379B1 (de) * 2015-06-26 2017-03-15 Technische Universität Graz Fusionsprotein
WO2019107896A1 (fr) * 2017-11-28 2019-06-06 울산과학기술원 Protéine de fusion comprenant une glutathion-s-transférase et une protéine ayant une affinité de liaison pour une cellule cible ou une protéine cible, et utilisation correspondante
US11844844B2 (en) 2017-11-28 2023-12-19 Unist(Ulsan National Institute Of Science And Technology) Fusion protein comprising glutathione-s-transferase and protein having binding affinity target cell or target protein, and use thereof

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