WO2010001414A1 - Expression de protéines hétérologues dans un système bactérien utilisant une étiquette de fusion gm-csf - Google Patents

Expression de protéines hétérologues dans un système bactérien utilisant une étiquette de fusion gm-csf Download PDF

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WO2010001414A1
WO2010001414A1 PCT/IN2009/000372 IN2009000372W WO2010001414A1 WO 2010001414 A1 WO2010001414 A1 WO 2010001414A1 IN 2009000372 W IN2009000372 W IN 2009000372W WO 2010001414 A1 WO2010001414 A1 WO 2010001414A1
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fusion
tag
terminus
fusion tag
fusion protein
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PCT/IN2009/000372
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Sampali Banerjee
Deshpande Anjali Apte
Naganath Mandi
Sriram Padmanabhan
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Lupin Limited
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/56—IFN-alpha
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the present invention is related to fusion tag comprising amino acids of human origin and having a very high potential to aid in expression of foreign genes. More particularly the invention relates to a fusion tag which comprises C-terminus domain of human Granulocyte-macrophage Colony Stimulating Factor (hGMCSF), START codon and cleavage enzyme site and adapted for expression of otherwise non- expressible genes.
  • the said tag is capable of expression of genes with rare codons in prokaryotic cells without supply of rare codons.
  • the present invention further relates to the said fusion tags being used in protein purification.
  • the present invention relates to expression vector comprising the fusion tag.
  • the present invention further relates to fusion protein comprising the fusion tag at its N terminus and nonGM peptide at the C terminus.
  • the invention also relates to method of formation of the vector and kit comprising the said fusion tag.
  • E.coli fusion proteins Three E.coli fusion proteins are identified BFR, GrpE and NusA that gave a high level of solubility when expressed as a fusion with target heterologous protein human interlukin-3 (hlL-3).
  • hlL-3 target heterologous protein human interlukin-3
  • Cabrita in his document teaches rationally designed T 7 based E coli expression vectors that include incorporation of solubility tags to assist in expression/purification, a TEV cleavable sequence and a LIC sequence. So, there are many commercial and non commercial E. coli expression vectors available that incorporate fusion tags (both for purification and enhanced solubility).
  • Literature evidences show that formation of secondary structures in transcribed mRNA reduces expression of heterologous genes. These secondary structures interfere with the binding of ribosome with mRNA thereby prevent efficient translation initiation. These deleterious secondary structures more likely occur due to short- range RNA-RNA interactions.
  • An N-terminal tag may provide a reliable context for efficient translation by reducing the possibility of secondary structure formation in mRNA and thus may improve the yield of recombinants proteins [Trends in Biotechnology (2005), 23, 316-320].
  • fusion tags might improve the yield of recombinant proteins by rendering them more resistant to intracellular proteases. Therefore, one approach to deal with this difficulty has been to express proteins as N- or C- terminal fusion tags.
  • the other tags available are thioredoxin (109 aa) [Biotechnology 11 187-193 1993], GST-tag (236 aa) [Gene 67 31-40 1988], MBP-tag (363 aa) [Eur J Biochem 171 541-549 1988], NusA (435 aa) [Biotechnol Bioeng 65 382-388 1999] etc.
  • thioredoxin 109 aa
  • GST-tag (236 aa) [Gene 67 31-40 1988]
  • MBP-tag (363 aa) [Eur J Biochem 171 541-549 1988]
  • NusA (435 aa) Biotechnol Bioeng 65 382-388 1999
  • the epitope tags commercially available. Often they are incorporated into the expression vectors for mammalian, insect, yeast or bacterial cells.
  • epitope tags are available, including c- myc, FLAG, HA, His6, T7-Tag, HSV-Tag, Pk-Tag, VSV-Tag, GIu-GIu, BTag and S- Tag.
  • Most epitopes that have been popular for the epitope tagging are highly charged (HA, c-myc, FLAG). Since- one generally aims to place the tag in the external portion of the target protein, it is appropriate that the tag be charged rather that hydrophobic. However, the tag of extreme or inappropriate charge could cause the problems in some cases, for example, if a basic domain of a protein is tagged with an acidic sequence.
  • the epitope tags without highly charged amino acids are T7-Tag and BTag.
  • the E2Tag consists of ten 10 amino acid residues, SSTSSDFRDR. US '895 designated this sequence as E2Tag. All these ten amino acids are required and are sufficient for strong interaction with monoclonal antibody 3F12.
  • the first half of the sequence consists of polar amino acids, and the second half contains charged amino acid residues resulting in very hydrophilic peptide.
  • Such highly hydrophilic sequences have the strong antigenicity and are correspondingly likely to adopt a highly exposed conformation in the three-dimensional folding of a protein.
  • tags are affinity tags and facilitates the purification of the fused proteins. Some of them (Trx, NusA etc.) are also reported to increase the solubility of the proteins when overexpressed [Harrison: Innovations, 11 :4-7 (2000)] and there are many commercial or non-commercial E.coli expression vectors available that incorporates fusion tags (for both purification and solubility). Most of these tags are large in size and of microbial origin.
  • Human Granulocyte macrophage colony stimulating factor is a glycoprotein growth factor which includes the proliferation of hematopoitic progenitor cells and functionally activates mature granulocytes and monocytes.
  • the processed human GM-CSF is a 127 amino acid polypeptide of which about 5-10 amino acids at the amino and the carboxy end are not essential while synthetic residues of 31 to 113 was biologically active. [Kanakura et. al., Blood 77 1033-1043 1991]
  • US 6379661 teaches novel polypeptides possessing part or all of the primary structural conformation and one or more of the biological properties of a mammalian (e.g., human) pluripotent GRANULOCYTE colony-stimulating factor ("hpG-CSF") which are characterized in preferred forms by being the product of prokaryotic or eukaryotic host expression of an exogenous DNA sequence. Sequences coding for part or all of the sequence of amino acid residues of hpG-CSF or for analogs thereof may be incorporated into autonomously replicating plasmid or viral vectors employed to transform or transfect suitable prokaryotic or eukaryotic host cells such as bacteria, yeast or vertebrate cells in culture.
  • suitable prokaryotic or eukaryotic host cells such as bacteria, yeast or vertebrate cells in culture.
  • Products of expression of the DNA sequences display, e.g., the physical and immunological properties and in vitro biological activities of isolates of hpG-CSF derived from natural sources.
  • Disclosed also are chemically synthesized polypeptides sharing the biochemical and immunological properties of hpG-CSF.
  • formation of the peptide of mammalian origin and its expression in bacterial host It shows formation of vectors with the DNA of GRANULOCYTE colony-stimulating factor ("hpG-CSF").
  • hpG-CSF GRANULOCYTE colony-stimulating factor
  • Example 4 in order to express the genes in the host, it requires the special codon like start codon to be present. Specifically it mentions that the sequence is not readily susceptible for securing direct expression of hpG-CSF in a microbial host and that to secure such expression, the hpG-CSF coding region should be provided with an initial ATG codon and the sequence should be inserted in a transformation vector at a site under control of a suitable promoter/-regulator DNA sequence. Accordingly though this art teaches that mammalian peptides could be expressed in microbial host, it requires specific start codons, in order to be expressed in the prokaryotic cells.
  • US 5359035 teaches bifunctional fusion protein comprising IL 2 and GM CSF which are linked together.
  • the said protein is capable of being expressed in bacteria.
  • the inventors have fused both IL2 and GMCSF together via a linker amino acid sequence.
  • both the proteins are complete and retain their respective biological activities.
  • few amino acids from the C terminus could be used to form a fusion protein, which would provide enhanced expression of otherwise non expressible genes.
  • EP1599589 (WO 2004/076670) teaches fusion proteins where tagging is carried out by biotinylation of the N terminal of peptides. This is used in fusion proteins and use of such tags increases the expression of genes in the host cells. This document though discloses enhancement of expression by use of fusion tags. However the said tagging is carried out by post translational manipulation i.e the fusion polypeptide needs to be biotinylated after synthesis for easy purification.
  • the GM fusion protein does not need any post-translational modification for purification. It is well known that the GM-CSF gene contains a heparin-binding domain and that domain lies at the C-terminus of the gene. The present inventors have found that this property can be exploited to use the said C terminus genes as fusion tag and purify the tagged proteins described here in a single step.
  • An object of the present invention is to provide a fusion tag comprising 45 amino acids of C terminus of the human GM CSF with a START codon and cleavage enzyme site and adapted to express otherwise non-expressible genes.
  • Another object is to provide a fusion tag such that it is capable of expression in a cell without requirement of rare codons.
  • a further object is to provide a fusion protein comprising fusion tag comprising 45 amino acids of C terminus of the human GM CSF with a START codon and cleavage enzyme site and adapted to express otherwise non-expressible genes and non GM protein.
  • Another object of the invention is to provide a vector comprising the fusion tag.
  • a further object is to provide a process for preparation of the fusion tag.
  • Another object is to provide a method of producing the fusion protein comprising the following steps: a) transforming a host cell with the DNA expression vector and b) expressing said fusion protein.
  • a further object is to provide a method of purifying protein with the fusion tag.
  • Another object is to provide a kit comprising the expression vector comprising the fusion tag.
  • An aspect of the present invention is to provide a fusion tag of 45 amino acids of the C terminus portion of human Granulocyte Macrophage Colony Stimulating Factor, START codon and cleavage enzyme site and adapted to express otherwise non expressible genes in prokaryotes.
  • a fusion protein comprising fusion tag towards the N terminus, said fusion tag comprising C terminus amino acids of human Granulocyte Macrophage Colony Simulating Factor (GM CSF), START codon and cleavage enzyme site and adapted to express other wise non expressible genes when cloned at C-terminus of the fusion tag.
  • GM CSF Granulocyte Macrophage Colony Simulating Factor
  • an expression vector comprising the fusion tag.
  • the vector may have one or more cloning sites.
  • a process for formation of the fusion tag comprising amplification of the C terminus 45 amino acids from a full length human GM-CSF synthetic gene using gene specific forward primers which contains specific restriction site for providing START codon and reverse primer containing sequences corresponding to cleavage enzyme site.
  • a method of producing the fusion protein comprising transforming a host cell with the DNA expression vector as defined above and expressing said fusion protein.
  • a method of purifying protein with the fusion tag comprising cloning the protein of interest to the expression vector, exposing to heparin sepharose, eluting the bound protein and ultimately obtaining the protein of interest bound to the heparin by the fusion tag which has affinity for binding to heparin.
  • kit comprising the expression vector comprising the fusion tag.
  • FIG. 1 Amplification of C terminus of GM CSF forming the Fusion tag (GM tag)
  • Figure 2 colony PCR of pET21 a-GM transformant
  • FIG. 3 Restriction digestion of two PCR positive clones- a new vector comprising fusion tag (GM tag) of present invention and named pCGM
  • Figure 7 SDS-PAGE analysis of rhlFN expression with or without GM tag in E. coli host BL21(DE3) Codon plus cells.
  • Figure 8 Expression of GM-/rhlFN in different E. coli expression hosts.
  • Figure 10 lmmunoblot analysis of GM-fusion protein (for example GM - GCSF fusion protein with antibodies against GMCSF and GCSF.
  • GM-fusion protein for example GM - GCSF fusion protein with antibodies against GMCSF and GCSF.
  • Figure 11 Cloning of C-term GMCSF into pET21a to construct pCGM (5.55 kbp)
  • Figure 12 Enterokinase Clevage site in commercial vectors
  • FIG. 13 Enterokinase Clevage site in the vector of present invention
  • C-terminus part of GM-CSF (the C-terminal part) is used as fusion tag to aid the expression of an otherwise non-expressible gene.
  • the portion of GM-CSF may or may not retain the biological activity of full length GMCSF.
  • the idea of using truncated GM-CSF as N-terminus fusion protein is hitherto unknown.
  • This domain of hGM-CSF has been found to have the properties to be used as fusion partner to achieve high level expression or to escape the requirement of rare codons for certain genes in a host prokaryotic, preferably bacterial host.
  • the C-terminus domain of hGM-CSF i.e the fusion tag, is located towards the N-terminus of the fusion protein and a non-GM peptide is located towards the C- terminus of the fusion protein.
  • the use of the antibody to the tag provides the rapid method for identifying, isolating, purifying and quantifying the amount of the fusion protein.
  • the antibody to the tag can be affixed onto sepharose or other beads suitable for the column separation.
  • the sample containing the chimeric protein is passed over the column containing the sepharose beads, or the binding is performed in batch by the end-over-end mixing, and the fusion protein having the tag is bound to the beads coupled by the specific antibody. It is followed by series of washes, the protein is eluted from the column using the standard elution techniques or the expression and/or the function of the fusion protein is studied by the matrix-attached tagged protein.
  • the tag of the invention is used to detect the protein expression in the transformed bacteria as well as in the transfected eukaryotic cells.
  • the tag is also used to identify the cellular localization of the fusion protein in the intact cell and to demonstrate the applicability of the tag for identifying the tracking of the protein through the cellular milieu by immunofluorescnence using commercially available anti hGMCSF antibody.
  • the fusion tag has an affinity to bind to heparin [Sebollela et. al., Journal of Biological Chemistry 280 31049-31956; 2005] and thus can be purified by affinity chromatography using immobilized heparin sepharose matrices.
  • fusion tag refers to the peptide having 45 amino acids of the C terminus of human GM CSF.
  • tagging refers to introducing by recombinant methods one or more nucleotide sequences encoding a peptide tag into a polypeptide encoding gene.
  • Fusion protein refers to the protein whose N terminus is formed by the fusion tag comprising the C terminus portion of human GM CSF and a non GM peptide at the C terminus.
  • This fusion tag contains cleavage enzyme site and has been designed in such a way that the fused peptide comprising the said tag can be obtained with no extra amino acid at N-terminus after enzyme cleavage.
  • the fusion tag has an affinity to bind to heparin [Sebollela et. al., Journal of Biological Chemistry 280 31049-31956; 2005] and thus can be purified by affinity chromatography using immobilized heparin sepharose matrices.
  • the fusion tag increases the expression of heterologous protein either by reducing the probability of formation of secondary structures near the ribosome binding site and thereby helps in efficient translation initiation or by increasing mRNA or protein stability.
  • the fusion tag has the following nucleotide sequence (SEQ ID 1)
  • the fusion tag does not require rare codons for certain genes like hi FN ⁇ 2b since the same cannot be expressed in ordinary host without supply of rare codons which are a prerequisite for interferon expression in E. coli [Annals New York Academy of Sciences; Olivares-Trjo et. Al, Molecular Microbiology, 2003, 49, 1043-1049].
  • the present fusion tag facilitates the expression of the gene containing rare codons either by altering the RNA secondary structure formation or by increasing the mRNA stability, without additional supply of tRNAs corresponding to rare codons.
  • Fusion tag of present invention increases the expression of heterologous protein either by reducing the probability of formation of secondary structures near the ribosome binding site and there by helps in efficient translation initiation or by increasing mRNA or protein stability.
  • the invention is related to the process for preparing heterologous proteins which are expressed by genes with rare codons in prokaryotic cells without supply of rare codons using the fusion tag of the invention.
  • genes with rare codons for expression in prokaryotes or having complex secondary mRNA structure at the N terminus region are but not limited to human lnterleukin 2 (hlL2), human interleukin 11 (hlL11); human granulocyte colony stimulating factor (hGCSF); human granulocyte macrophage colony stimulating factor (hGMCSF); rPA: human reteplase (rPA); human epidermal growth factor (hEGF); human platelet derived growth factor (hPDGF); human growth hormone (hGH); human interleukin 3 (hlL3); human interferon gamma (hlFN ⁇ ; human interferon alpha 2a (hlFN ⁇ 2a).
  • the invention is related to the heterologous protein obtained by a process comprising expression of genes with rare codons in prokaryotic cells without supply of rare codons using the fusion tag of the invention.
  • the GM fusion tag is provided with a START codon and any gene can be cloned at the C-terminus of GM tag that may or may not contain ATG depending on the sequence of the mature peptide and the tag is capable of expressing.
  • an expression vector comprising a DNA sequence coding for a fusion protein said fusion protein comprising a fusion tag being capable of being used in N- terminus tagging, wherein the fusion tag has the 45 amino acid of human GM CSF with a STRT codon and cleavage enzyme site.
  • a gene of interest to be tagged with said fusion tag expressing protein of interest directly cleavable from cleavage site without intervening amino acids.
  • the vector is such that the gene of interest is expressed in higher amount which was otherwise non- expressible without a tag.
  • the vector is such that the gene of interest is expressed without supplement of START codons (if mature peptide does not have methionine as first amino acid) in the host unlike other vectors known in the art.
  • Such vector designated as pCGM may be constructed and made available to public. Further, the fusion construct (i.e. vector pCGM) could be made available commercially.
  • cleavage enzyme sites can be used in the invention.
  • cleavage enzyme sites are but not limited to enterokinase, caspases, thrombin, factor Xa, TEV protease, KEX proteases sites and so on.
  • the preferred cleavage enzyme site is enterokinase.
  • the proteins obtained using the fusion tag of the invention possess bioactivity after refolding. There is an efficient cleavage of the fusion protein using cleavage enzyme indicating that the fusion tag not only helps in expression of difficult proteins, it actually helps it to possibly express in the right confirmation also.
  • Specific primers are designed to amplify the C-terminus GM-CSF from a synthetic gene construct corresponding to human GMCSF as described in Example"! .
  • the forward primer contains sequence corresponding to Ndel restriction site. This is capable to incorporate the START codon in the fusion tag.
  • the reverse primer is designed to have BamHI restriction site followed by a stretch of sequences that code for cleavage enzyme site and then an EcoRI site.
  • the cleavage enzyme site is incorporated in the fusion tag and the fusion tag comprising the 45 amino acid of the C terminus of hGMCSF, START codon and the cleavage enzyme site is formed. This is illustrated in Figure 11.
  • FIG. 11 Schematic presentation of the vector construct with GM -tag (I) containing enterokinase (II) cleavage site is shown in figure 11.
  • Commercially available expression vector is digested with specific enzymes and ligated to the fusion tag of the present invention.
  • the ligation mix is introduced into appropriate E. coli host by a method known as transformation.
  • Gene of interest (GOI, III) is expressed as N- terminus GM fusion and could be cleaved by enterokinase to obtain protein of interest without any extra amino acid at N -terminus since the tag does not have any additional amino acid at this end.
  • the gene of interest (GOI), to be cloned as the C terminus protein and the GM fusion tag of present invention forms the N terminus, is PCR amplified using gene specific 5'primer containing BamHI restriction site followed by sequence corresponding to EK site and 3' primer containing EcoRI site (see example 2). This amplicon is cloned into pCGM at BamHI/EcoRI sites.
  • the vector with fusion tag is such that no extra amino acids at the N terminus of the synthesized protein after enzyme cleavage. This vector with the fusion tag is capable of being used to clone any foreign genes.
  • fusion protein contains at least two extra amino acids (Glycine and Serine) as encoded by nucleotide SEQ ID 3 GAT GAT GAT GAT AAA GGA TCC ATG and having amino acid sequence SEQ ID4 D D D D K G G M out of which enetrokinase cleavage site is formed of D D D D K and the BamHI site with G as described in figure 12.
  • the vector comprising the fusion protein of the present invention as encoded by nucleotide SEQ ID 5 GGA TCC GAT GAT GAT GAT AAA ATG and having amino acid sequence SEQ ID6 G S D D D D K M out of which enetrokinase cleavage site is formed of D D D D K and the Bam site with G and S as described in figure 13.
  • a further aspect is to provide a method of producing a fusion protein comprising transforming a host cell with the DNA expression vector as defined above and expressing said fusion protein.
  • the host cell is prokaryotic cell, preferably an E. coli.
  • the method of identifying, purifying or isolation of the fusion protein may be carried out by using an antibody raised against the fusion tag (more precisely, against full length hGMCSF) using immunoblotting to identify the tagged protein of interest.
  • the kit for the protein tagging comprises reagents like Heparin sepharose (immobilized column material) for tagged protein purification specific for the peptide tag of the invention.
  • the kit comprise additionally the DNA expression vector comprising DNA coding for the peptide tag of the invention.
  • the kit comprises the expression vector comprising the sequences coding for the peptide tag of the invention and having at least one cloning site. The vector may have multiple cloning sites.
  • the peptide/ protein of interest/ to be purified is cloned with the expression vector. It is then run over the heparin sepharose. In view of the affinity for the heparin the fusion tags will bind to it and along with it the protein if interest will also be bound. This would facilitate separation/ purification of proteins using the fusion tag of the present invention.
  • GM fusion tag capable of expression of genes containing rare codons, in prokaryotes without additional rare codon specific tRNA supplementation. Heparin binding domain for affinity purification. The tag could be easily cleaved off by enzyme to get authentic N terminal of the protein of interest. It could be easily detected by commercially available anti-h GM-CSF antibody. Tagged fusion protein could be more active than the native protein and may be of potential use in diagnostics. The tag could be less immunogenic as it is of human origin. Since the tag is small, there is a possibility of the fusion protein being more protease resistant.
  • GM tag is the 45 amino acids long C-terminus part of human GM-CSF gene.
  • the tag was amplified from a full length human GM-CSF synthetic gene using gene specific primers as mentioned below.
  • the forward primer was designed in such a way that it contains an Ndel restriction site (which provides a START codon (ATG) at the N-terminus of the tag).
  • the reverse primer contains two restriction sites, namely EcoRI and BamHI to enable the construction of the vector and subsequent cloning of the genes of interest. Sequences corresponding to cleavage enzyme site have been incorporated in the reverse primer flanking between the two above mentioned restriction sites.
  • Reverse primer 5 1 ccg ccg gaa ttc ttt ate ate ate ate gga tec gac tgg etc cca gca gtc 3'. (SEQ ID 8)
  • the GM tag was amplified using PCR, the amplicon was purified using gel extraction kit from Sigma following manufacturer's protocol and digested with Ndel and EcoRI (Fig 1). The digested amplicon is ready to use for construction of the vector.
  • the gene corresponding to the protein is amplified using specific primers that contains BamHI site followed by EK site in the forward primer and and EcoRI in the reverse primer.
  • the PCR amplified product the digested with same set of enzymes and ligated to pCGM as BamHI/EcoRI fragment.
  • the present example is aimed at cloning hlFN ⁇ 2b as N-terminal GM fusion and investigate the requirement of rare codon of GM-hlFN ⁇ 2b while expressing in E. coli.
  • the hlFN ⁇ 2b gene was amplified with specific primers using synthetic gene as template (fig. 4).
  • the amplicon was digested and ligated to the pCGM vector at BamHI/EcoRI site that contains GM tag peptide as N-terminal fusion including EK site.
  • the clones were screened by colony PCR using same set of primers (fig. 5)
  • the PCR positive clones were confirmed by restriction digestion with BamHI/ EcoRI and Ndel/ Hindlll that would release ⁇ 500bp and ⁇ 638 bp, respectively (fig. 6).
  • GM tag without any foreign gene protein was taken as control.
  • GM-hlFN clones as formed in example 2 were introduced into E. coli expression host BL21 (DE3) codon Plus for expression studies. HIFN ⁇ 2b clone without GM fusion was used as control. The cells were induced with 1mM IPTG for expression of IFN ⁇ 2b and induction was continued for 4 hours at 37°C. After induction, soluble and insoluble fractions were separated and analysed on SDS PAGE (fig. 7).
  • Yield of rhlFN production is ⁇ 20% higher when expressed as GM-fusion compared to expression without fusion (fig. 7, right panel lanes: GM-IFN and 21 ⁇ -IFN, respectively).
  • insoluble protein could be made soluble when tagged with the fusion peptide in the fusion protein of the present invention.
  • IFN ⁇ 2b expression in different E. coli hosts IFN ⁇ 2b was expressed as native and tagged fusion in three different E. coli hosts. As evident from the fig. 8 (Lane: 1, 2, 3 : Native IFN in BL21A1, BL21(DE3) and BL21(DE3) codon+ respectively. Lane: 4, 5, 6: GM IFN fusion in BL21A1 , BL21(DE3) and BL21(DE3) codon+ respectively), IFN does not express in hosts, which do not supplement rare codons (lanes land 2).
  • GM fusion facilitates expression of those proteins that are otherwise difficult to express
  • Clones of untagged hlL11 and hlL2 did not express in tested bacterial hosts, like BL21A1 , BL21 DE3, BL21 DE3 Codon Plus, etc.(data not shown).
  • the fusion protein with the GM fusion tag is used to express these proteins.
  • Both hlL11 and IL2 were cloned as GM fusion peptide similarly as IFN ⁇ 2b and introduced into E. coli host BL21A1 for expression studies and cells were induced with 13mM Arabinose at 37°C.
  • GCSF is cloned in pCGM vector as GM-GCSF fusion as in example 1 and expression was carried out in E. coli expression host BL21A1 as before.
  • the immunoblot analysis was done with both antiGCSF and antiGMCSF antibodies.
  • Fig. 10 shows that GM-GCSF is recognised by GCSF antibody (lanei) as well as by

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Abstract

L'invention concerne une étiquette de fusion comportant des acides aminés de terminaison C du facteur de stimulation humain de colonies de macrophages et de granulocytes (GM-CSF), un codon START et un site de clivage à l'entérokinase et qui est adaptée pour exprimer d'autres gènes wise non exprimables. L'invention concerne également un procédé de formation de l'étiquette de fusion qui comprend l'amplification des acides aminés 45 de terminaison C à partir d'un gène synthétique de GM-CSF humain pleine longueur utilisant des amorces avant spécifiques au gène qui contient un site de restriction spécifique destiné à fournir le codon START, et des séquences contenant une amorce inverse correspondant au site de clivage à l'entérokinase, une protéine de fusion comportant l'étiquette de fusion en direction de la terminaison N, ladite étiquette de fusion comportant des acides aminés à terminaison C du facteur de stimulation humain de colonies de macrophages et de granulocytes (GM-CSF), un codon START et un site de clivage à l'entérokinase et étant adaptée pour exprimer d'autres gènes wise non exprimables, et un peptide non-GM en direction de la terminaison C. L'invention concerne en outre un procédé de production de la protéine de fusion qui comprend la transformation d'une cellule hôte avec un vecteur d'expression d'ADN tel que défini dans et exprimant ladite protéine de fusion. L'invention concerne également un procédé de purification ou d'isolement de la protéine de fusion. De plus, l'invention concerne un vecteur d'expression comportant une séquence d'ADN codant une protéine de fusion, ladite protéine de fusion comprenant une étiquette de fusion pouvant être utilisée dans le marquage de la terminaison N, ladite étiquette de fusion comportant l'acide aminé 45 du facteur humain GM-CSF, un codon START et un site de clivage à l'entérokinase.
PCT/IN2009/000372 2008-07-03 2009-07-02 Expression de protéines hétérologues dans un système bactérien utilisant une étiquette de fusion gm-csf WO2010001414A1 (fr)

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IN1164/KOL/2008 2008-07-03
IN1164KO2008 2008-07-03

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WO2013076657A1 (fr) * 2011-11-22 2013-05-30 Lupin Limited Protéine hybride map
WO2014142453A1 (fr) * 2013-03-14 2014-09-18 전남대학교산학협력단 Etiquette de surexpression protéique universelle comprenant une fonction ramp, et son application
WO2015112121A1 (fr) * 2014-01-21 2015-07-30 The Board Of Trustees Of The University Of Arkansas Étiquette d'affinité de l'héparine utile dans la purification des protéines
EP3373954A4 (fr) * 2015-11-10 2019-07-03 Proteothera, Inc. Procédés de production et de purification de protéines de fusion se liant à une matrice par chromatographie par échange d'ions

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013076657A1 (fr) * 2011-11-22 2013-05-30 Lupin Limited Protéine hybride map
WO2014142453A1 (fr) * 2013-03-14 2014-09-18 전남대학교산학협력단 Etiquette de surexpression protéique universelle comprenant une fonction ramp, et son application
KR101446054B1 (ko) 2013-03-14 2014-10-01 전남대학교산학협력단 재조합 단백질의 과발현을 위한 번역속도 조절용 램프 태그 및 이용
CN105143464A (zh) * 2013-03-14 2015-12-09 全南大学校产学协力团 包含ramp功能的通用蛋白质过表达标记及其应用
CN105143464B (zh) * 2013-03-14 2017-06-30 全南大学校产学协力团 包含ramp功能的通用蛋白质过表达标记及其应用
US10227595B2 (en) 2013-03-14 2019-03-12 Industry Foundation Of Chonnam National University Universal protein overexpression tag comprising ramp function, and application thereof
WO2015112121A1 (fr) * 2014-01-21 2015-07-30 The Board Of Trustees Of The University Of Arkansas Étiquette d'affinité de l'héparine utile dans la purification des protéines
WO2015112507A1 (fr) * 2014-01-21 2015-07-30 The Board Of Trustees Of The University Of Arkansas Marqueur d'affinité pour l'héparine et applications correspondantes
US9676816B2 (en) 2014-01-21 2017-06-13 Board Of Trustees Of The University Of Arkansas Heparin affinity tag and applications thereof
US10280196B2 (en) 2014-01-21 2019-05-07 The Board Of Trustees Of The University Of Arkansas Heparin affinity tag and application thereof
EP3373954A4 (fr) * 2015-11-10 2019-07-03 Proteothera, Inc. Procédés de production et de purification de protéines de fusion se liant à une matrice par chromatographie par échange d'ions

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