The present invention relates to a method for the production of high-level soluble human recombinant interferon alpha (rhuIFNα) in E. coli and vectors useful for such a production.
Interferons (IFNs) are a group of naturally-produced glycoproteins endowed with antiviral, anti-proliferative, and immuno-modulatory properties (Pfeffer, 1997; Pestka et al., 1987) as well as an analgesic action (Wang et al., 2000; Wang et al., 2002). The medical potential of IFNs was soon recognized, as demonstrated by the approval in 1986 of recombinant human IFNα2a (Roferon-A), and later IFNα2b (Intron-A), as drugs for the treatment of malignant and viral diseases (Gutterman et al., 1994; Lauer et al., 2001; Motzer et al., 2002; Mahon et al., 2002).
Most of the marketed pharmaceutical grade recombinant IFNα has since been produced and purified from E. coli.
The E. coli recombinant protein expression system has been, and still is, the system of choice for the production of IFNα. Indeed, IFNα genes do not have introns, and the protein products are generally not glycosylated. Furthermore, E. coli can grow rapidly to high cell densities, and strains used for recombinant protein production have been genetically modified so that they are generally regarded as safe for large-scale fermentation.
The expression of IFNα cDNA was achieved directly in E. coli soon after it was first cloned (Nagata et al., 1980; Pestka et al., 1983; Goeddel et al. 1980; Mizoguchi et al., 1985; reviewed by Pestka et al., 1987; reviewed by Barron and Narula 1990).
Several promoter systems were chosen to achieve high intracellular expression levels (Laplace et al., 1988; Boyer et al., 1992; Swaminathan et al., 1999; Babu et al., 2000; Lim et al., 2000; Bedarrain et al., 2001; Neves et al., 2004, Srivasta et al. 2005).
However, IFNα protein expressed in large amount in E. coli often precipitate into insoluble aggregates called inclusion bodies (Swaminathan et al., 1999; Bedarrain et al., 2001; Srivasta et al. 2005) that are, in general, misfolded proteins and thus biologically inactive (Villayerde and Carrio, 2003).
In many cases, refolding from inclusion bodies (Middelberg, 2002) is considered undesirable, because of the poor recovery yield and the requirement for optimization of the refolding conditions for each target protein. Furthermore, resolubilization procedures may not fully restore the folding of the protein, and therefore its optimal function.
Solubility is a key issue for the production of recombinant protein in heterologous expression systems. Soluble recombinant proteins are often properly folded, functional, and easier to purify than aggregated proteins from inclusion bodies.
Hence, maximizing the production of recombinant proteins in a soluble form is an attractive alternative to the in vitro refolding procedures. Furthermore, it has been shown that fusion proteins have the advantage of providing a more favourable gene construct organization, permitting high levels of soluble protein to be expressed (Kapust and Waugh, 1999) by reducing the propensity to drive the protein folding process towards creating inclusion bodies (Lilie et al., 1998).
Two main approaches are generally used, separately or in combination, to favour expression of soluble recombinant proteins.
The use of low temperature, for instance, has the combined advantages of slowing down transcription and translation rates and of reducing the strength of hydrophobic interactions that contribute to protein misfolding. However, the drawback of this approach (as in the case with low inducer concentrations) is a reduction in productivity and
The separation of the recombinant target protein from the affinity tag or solubility enhancers, is achieved by site-specific proteolysis; two serine proteases, namely factor Xa and thrombin are usually extensively employed. However selection of optimal reaction conditions and a specific protease depends on the recombinant target protein.
Several other expression systems were used to overcome the problem of inclusion bodies and improve protein solubility for the expression of human recombinant IFNα. These include Bacillus subtilis (Palva et al., 1983), Streptomyces lividans (Pulido et al., 1986), methylotrophic yeasts such as Pichia pastoris (Hitzeman et al., 1981; Tuite et al., 1982; Liu et al., 2001), Murine Myeloma NSo cells (Rossmann et al., 1996), and baculovirus-infected insect cells (Maeda et al., 1985). Most of these systems have allowed the expression of soluble human recombinant IFNα; however none of them allowed the yields obtained in E. coli within inclusion bodies.
Thus, although the production of recombinant proteins in E. coli is well established, there are numerous factors which may present obstacles for successful production and purification of soluble recombinant proteins (Baneyx and Mujacic, 2004), the main obstacle being inclusion body formation and proteolytic degradation.
As regards interferon alpha 2, the highest amounts of recovery are reported in Srivasta P. et al., 2005, in which the refolding and purification yield were found to be ˜3 g/l with 58% recovery. The conditions were the following: recombinant E. coli D115α cells containing a plasmid expressing IFNα (pRSET-IFNα) which had the IFN-α2b gene under the T7 promoter was coexpressed with a plasmid which carried the gene for T7 RNA polymerase under the heat inducible λPL promoter. This two plasmids expression system was optimized with respect to heat shock time, media and time of induction in a first step; the IBs (inclusion bodies) represented ˜40% of the total cellular protein; in a second step the IBs were isolated and purified through ion exchange followed by step refolding to give a final product yield of ˜3 g/l. This procedure constitutes a laborious procedure.
All the methods described in the prior art either are not adapted to interferon alpha or lead to low yields of interferon alpha.
Even though both Srivasta et al., 2005 and Babu et al., 2000 propose methods of solubilization and purification of IFNα from inclusions bodies (IBs), with a good yield, these methods are time consuming and costly.
The purification of soluble recombinant proteins is more cost effective and less time consuming than refolding and purification from inclusion bodies.
Accordingly, there remains a need for a method that enables the production of interferon alpha from bacterial cells in a high yielding and cost-effective manner.
The Inventors have developed a strategy to drive the expression of human IFN α and more precisely IFN α2b in E. coli from aggregated protein in the inclusion bodies to soluble and properly folded cytoplasmic protein.
In the context of the invention, the Inventors have surprisingly found that production of soluble recombinant human IFN alpha can be achieved in E. coli by using a fusion protein GST-IFNα; this solution represents a more economical and efficient method for preparing IFN alpha than the ones proposed in the here above cited documents.
Therefore, a first aspect of the invention relates to a method for preparing a recombinant interferon alpha protein by expression in E. coli, said method being characterized in that it comprises the steps of:
(1) Transforming a E. coli strain selected in the group consisting of E. coli protease deficient host strains and E. coli reductase deficient host strains with a recombinant expression vector comprising the sequence encoding the glutathione-S-transferase, a junction sequence including a recognition site for a specific protease and a sequence able to encode an interferon alpha (IFN alpha or IFN α) protein under the control of an inducible promoter, said vector encoding therefore a GST-IFN α protein,
(2) Expressing said interferon alpha protein in conditions comprising the induction of the expression with 0.1 mM-0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and a growth temperature of 25° and/or 37° C., depending on said E. coli strain and
(3) Isolating the expressed IFN alpha protein.
According to a first advantageous mode of carrying out the method of the invention, the junction sequence of said vector consists of the sequence CTG GTT CCG Z1 TCC Z2, wherein Z1 represents a thrombin recognition site CGT GGM and Z2 represents CCG GAA TTC TGT (SEQ ID NO: 3) or TGT; therefore, when Z2 represents SEQ ID NO:3, the junction sequence is represented by SEQ ID NO: 1 and when Z2 represents TGT, the junction sequence is represented by SEQ ID NO: 2.
Two preferred following vectors may thus be obtained:
According to a second advantageous mode of carrying out the method of the invention, the E. coli protease deficient strain is an E. coli lon−/ompT− protease deficient host strain, preferably an E. coli BL21 strain, deposited at the CNCM (Collection Nationale de Culture de Microorganismes, 28 rue du Docteur Roux, 75015 PARIS) on Jun. 1, 2007 under the accession number I-3769.
According to a third advantageous mode of carrying out the method of the invention, the E. coli reductase deficient host strain is an E. coli trxB−/gor− reductase deficient host strains, preferably an E. coli Origami B strain DF5Δ/INF/IPT06, deposited at the CNCM (Collection Nationale de Culture de Microorganismes, 28 rue du Docteur Roux, 75015 PARIS) on Apr. 30, 2007 under the accession number I-3760.
Surprisingly, the here above conditions lead to an enhancement (up to 70%; e.g., 1, 5, 10, 25, 30, 40, 50, 60, 65, 69, or 70 or any intermediate value or subrange) of the expression of IFNα as an intracellular soluble fusion protein in both E. coli strains.
However, in BL21 strain, this result was achieved only at a growth temperature of 25° C. and induction with 0.1 mM-0.5 mM IPTG, whereas in Origami B cells, this result was achieved both at 25° C. and at 37° C.
More precisely:
Thus, according to a fourth advantageous mode of carrying out the invention, when the E. coli strain is an E. coli BL21 strain, the conditions of step (2) comprise a growth at 25° C. and induction with 0.1-0.5 mM IPTG.
According to a fifth advantageous mode of carrying out the method of the invention, when the E. coli strain is an E. coli Origami B strain, the conditions of step (2) depend on the vector:
High cell density fed-batch culture conditions of E. coli are for instance described in Yee L et al., 1992.
According to another advantageous mode of carrying out the method of the invention, said inducible promoter is the tac promoter. Other inducible promoters usable in E. coli may also be used, such as for instance the λPL promoter.
According to yet another advantageous mode of carrying out the method of the invention, step (3) of isolating the expressed IFN alpha protein comprises successively, after lysis of the E. coli cells, centrifugation and retrieval of the supernatant:
IFN alpha proteins are described for instance in Baron E. et al.; U.S. Pat. No. 5,710,027 and Nyman T. A. et al.; database accession numbers of the main IFN alpha proteins are the following: NM—024013; NM—000605; V00549; AY255838; NM—021068; NM—002169; NM—021002; NM—021057; NM—002170; NM—002171; NM—006900; NM—002172; NM—002173; NM—021268; NM—002175.
IFN alpha proteins which may be produced according to the instant invention may be any of the hereabove mentioned IFN alpha proteins or IFN alpha proteins having at least 70%, 80%, 90%, 95%, 99% identity with one of the cited IFN alpha protein.
X % of identity between an IFN alpha protein P and a IFN alpha protein of reference R, means that when the two sequences are aligned, X % of the amino acids of P are identical to the corresponding amino acid in sequence R or are replaced by an amino acid of the same group:
Amino Acids with Nonpolar R Groups
Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine.
Amino Acids with Uncharged Polar R Groups
Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine.
Amino Acids with Charged Polar R Groups (Negatively Charged at pH 6.0)
Aspartic acid, Glutamic acid.
Basic Amino Acids (Positively Charged at pH 6.0)
Lysine, Arginine, Histidine (at pH 6.0).
Another grouping may be those amino acids with phenyl groups:
Phenylalanine, Tryptophan, Tyrosine.
Another grouping may be according to molecular weight (i.e., size of R groups):
Particularly preferred conservative substitutions are:
These percentages of sequence identity may be obtained using the BLAST program (blast2seq, default parameters) (Tatutsova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250).
Surprisingly, the modifications of the expression plasmid (engineering of the GST-IFN junction including codon optimization) as well as the use of the modified E. coli expression strain trxB−/gor− double mutant, Origami B, allowed the production of 100 mg/L of pure, soluble, and functional recombinant hIFNα.
Therefore according to a sixth mode of carrying out the method of the invention, at least 100 mg/L of soluble IFN alpha is obtained. More than 300 mg/L may be obtained in high cell density fed-batch cultures of E. coli.
According to another mode of carrying out the invention, the sequence encoding said interferon α protein is the sequence encoding interferon α2 protein. Said sequence encoding said interferon α2 protein comprises preferably the sequence encoding IFN α2b protein or a sequence encoding an interferon α2 protein which has more than about 70%, 80%, 90%, 95% or 99% identity with the IFN alpha2 protein of SEQ ID NO: 15.
The invention also relates, in a second aspect, to a vector for expressing soluble interferon alpha in E. coli, characterized in that it comprises the sequence encoding the glutathione-S-transferase (GST), a junction sequence including a recognition site for a specific protease and a sequence able to encode an interferon alpha (IFN alpha) protein.
According to a first embodiment of carrying out said vector, the junction sequence of said vector consists of the sequence CTG GTT CCG Z1 TCC Z2, wherein Z1 represents a thrombin recognition site CGT GGM and Z2 represents CCG GAA TTC TGT (SEQ ID NO: 3) or TGT (SEQ ID NO: 1 and SEQ ID NO: 2).
According to one mode of carrying out said embodiment, the junction sequence consists of the sequence CTG GTT CCG CGT GGA TCC CCG GAA TTC TGT (SEQ ID NO: 4) (Z1=CGT GGM, with M=A and Z2=CCG GAA TTC TGT (SEQ ID NO: 3)).
According to another mode of carrying out said embodiment, the junction sequence consists of the sequence CTG GTT CCG CGT GGC TCC TGT (SEQ ID NO: 5) (Z1=CGT GGM with M=C and Z2=TGT).
According to a second embodiment of carrying out said vector, the sequence encoding said interferon α protein is a sequence encoding interferon α2. Said sequence encoding said interferon α2 protein comprises the sequence encoding IFN α2b protein.
The sequence of IFN alpha proteins are as defined hereabove.
Besides the above provisions, the invention also comprises other provisions which would emerge from the following description, which refers to examples of implementation of the invention and also to the attached drawings, in which:
The following examples illustrate the invention but in no way limit it.
Strains
The E. coli JM109/recA−, endA− strain (Stratagene) was used as the host strain for routine cloning experiments. The E. coli trxB−/gor− deficient strain, Origami B (Novagen, Madison, Wis.), and the E. coli lon− and ompT− BL21 protease-deficient strains (Amersham Pharmacia Biotech), were used as host strains for recombinant GST-Δ-huIFNα2b expression.
Construction of Recombinant pGEX-huIFNα2b Expression Vector
Human Interferon α2b cDNA was cloned by an RT-PCR approach using mRNA prepared from healthy individual leukocytes exposed in vitro to the Newcastle disease virus as described by Wheelock et al. 1966 and Waldmann et al. 1981, using the TRIZOL™ mRNA extraction method as described by the manufacturer (Invitrogen). The cDNA corresponding to the IFNα2b published sequence (Pestka, 1983) was amplified using a forward primer that introduced an EcoRI site at the 5′ end of the gene (5′-TGGAATTCTGTGATCTGCCTCAAACCCA-3′ (SEQ ID NO:10)) and a reverse primer containing the XhoI site at the 3′ end of the gene (5′-CGCTCGAGTCATTCCTTACTTCTTAAACTTTC-3′ (SEQ ID NO:11)). The purified PCR product was digested with EcoRI and XhoI restriction enzymes and inserted into the plasmid pGEX4T1 (Amersham Biosciences) to generate the pGEX-huIFNα2b expression vector. Screening of pGEX4T1/IFNα2b recombinant plasmids containing the cDNA sequence encoding human IFNα2b was performed by a restriction mapping analysis using BglII restriction enzyme as recommended by the manufacturer (Amersham Biosciences). Finally, the nucleotide sequence of the selected clones was checked by automated DNA Sequencing Analysis using the “ABI-PRISM377” DNA sequencer (Perkin Elmer Applied Biosystems). The 5′ pGEX sequencing primer (Amersham Biosciences) was used as the sequencing primer.
Construction of Recombinant pGEX-Δ-huIFNα2b Expression Vector
The pGEX-huIFNα2b expression vector was used as the DNA template for site-directed mutagenesis (PCR-SDM) procedures as described by Rabhi et al. 2004, using a pair of mutagenic primers (Genset-Oligos/Paris, France) as described in
Analytical Expression of Recombinant GST-rhIFNα2b
The Origami B and BL21 E. coli cell lines were transformed with the wild type pGEX-rhuIFNα2b plasmid and the GST-IFN junction reengineered pGEX-Δ-hIFNα2b plasmid, using the TSS method following standard protocols.
Starter cultures of 5 ml LB (Luria-Bertani) medium containing 100 μg/ml ampicillin were inoculated, each with a single E. coli Origami B or BL21 recombinant clone. The cultures were grown overnight at 250 rpm and 37° C. 1 ml of the overnight culture was added to 100 ml LB medium supplemented with 100 μg/ml ampicillin and further incubated at 37° C. up to an OD600 of 0.5.
Monitoring of Growth Conditions, i.e. Temperature and IPTG Concentration, Using E. coli BL21 and Origami B Strains
To check the effects of the inducer (IPTG) concentration and culture growth temperature on the expression of soluble GST-hIFNα2b wild type and GST-Δ-hIFNα2b mutant recombinant proteins, each host strain culture was induced with three IPTG concentrations (0.1, 0.5, and 1 mM) at an OD600 of 0.5, and at temperatures of 25° C. and 37° C. until reaching an OD at 600 nm of 2.
Extraction of GST-huIFNα2b Recombinant Protein
Cells from induced and uninduced cultures were harvested by centrifugation (4000 g, 30 min, 4° C.) followed by two washing steps with buffer A (10 mM Na2HPO4, 1.8 mM KH2PO4, 140 mM NaCl, 2.7 mM KCl, pH 7.3) at 4000 g for 30 min, and finally stored at −70° C. until use. Protein extraction was performed by resuspending the cell pellet in ⅕ of the original culture volume of buffer B (10 mM Na2HPO4, 1.8 mM KH2PO4, 140 mM NaCl, 2.7 mM KCl, pH 7.3, and 1% Triton X-100). The cells were disturbed by six 30 second sonication steps. The supernatant was collected by centrifugation at 4° C. for 30 min at 13500 rpm and stored for GST-Δ-huIFNα2b expression analysis. Finally, cell pellets corresponding to insoluble protein fractions (such as inclusion bodies) were washed separately with the same volume of buffer B.
Recombinant Protein Expression Analysis
To analyze the intracellular expression of GST-Δ-huIFNα2b recombinant fusion protein in E. coli host cells, the clear supernatants were subjected to SDS-PAGE. Electrophoresis was performed using 15% SDS-polyacrylamide gels stained with Coomassie Brilliant Blue as described by Laemmli.
The recombinant fusion protein was detected by Western blot-ECL Assays (Amersham Biosciences) performed according to the manufacturer's instructions using either anti-GST peroxidase-conjugated sheep antibody at a dilution of 1:10,000 (Amersham Biosciences) or 1:400 dilution of Anti-human IFNα polyclonal antibody (ENDOGEN Searchlight), followed by the anti goat/sheep IgG peroxidase-conjugated monoclonal antibody (Sigma) used as the second antibody.
The ImageJ software was used to compare fusion protein expression under different growth conditions (e.g. IPTG concentration and growth temperature).
The concentration of GST-Δ-hIFNα2b in Origami B lysate versus rhuIFNα2b obtained after thrombin cleavage and the two-step purification was also determined by a quantitative in-house-developed ELISA assay. Dilution series containing 0 to 570 pg of HPLC-purified soluble IFNα2b produced in our laboratory were included in each assay to construct a standard curve. Recombinant proteins were detected using anti-human IFNα biotin-labelled monoclonal antibody (ENDOGEN Searchlight) and a colorimetric detection system using a streptavidin-horseradish peroxidase (HRP) conjugate (Amersham Biosciences).
The purity of the recombinant huIFNα2b was checked by analysis of 5 μg recombinant protein on Coomassie-blue and silver-stained SDS-PAGE 15% gels.
Affinity Chromatography Step and Thrombin Cleavage of GST-hIFNα2b Recombinant Protein
The supernatant containing the soluble GST-hIFNα2b recombinant protein was loaded on a GSTrap FF affinity column (1 ml; Amersham Biosciences) pre-equilibrated with buffer A (10 mM Na2HPO4, 1.8 mM KH2PO4, 140 mM NaCl, 2.7 mM KCl, pH 7.3) at a flow rate of 1 ml/min at room temperature. The bound material was washed with buffer A until the absorbance at OD280 nm returned to base line. Once the baseline was stable, elution of the bound GST-Δ-huIFNα2b recombinant protein was carried out using 6 column volumes of elution buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0) at a 0.5 ml/min flow rate. The eluted fractions containing the GST-huIFNα2b recombinant protein were pooled. The purification stages and affinity chromatographic profiles were analyzed by Coomassie blue-stained SDS-PAGE gels and by Western blot analysis as described above.
Twenty units of thrombin solution were added to 100 μg of eluted fusion protein and incubated at room temperature (+22° C.) for 20 hours.
Size Exclusion Purification Step of hIFNα2b Recombinant Protein
Upon completion of thrombin digestion, the glutathione and thrombin were removed by a size exclusion chromatographic step. The digested product was loaded on a size exclusion Sephacryl S-100 26/60 High Resolution column (Amersham-Biosciences), and the cleaved rhuIFNα2b peak was eluted. The chromatographic profile was evaluated by Coomassie blue and silver-stained SDS-PAGE gels.
Biological Activity of rhIFNα2b
The biological activity of the recombinant huIFNα2b preparation was determined by the antiviral and Gene Report assays as described by A. Meager, 2002, at the Division of Immunobiology, National Institute for Biological Standards and Control, UK. One unit of activity was defined as the amount of recombinant hIFNα2b required to produce antiviral activity equivalent to that expressed by 1 IU hIFNα2b reference standard (code: 95/566; Division of Immunobiology; National Institute for Biological Standards and Control, Potters Bar, UK).
Clone stability was checked after six months of continuous culture by plasmid DNA preparation and DNA sequencing of the expression cassette.
Human Interferon α2b cDNA was cloned by an RT-PCR approach using mRNA prepared from the leukocytes of a healthy individual that were exposed in vitro to the Newcastle disease virus. The cDNA corresponding to the published sequence [38] was amplified using a forward primer that introduces an EcoRI site at the 5′ end of the gene and a reverse primer containing the XhoI site at the 3′ end of the gene. The purified PCR product was cloned between the EcoRI 5′ end and XhoI 3′ end of pGEX4T1, downstream of the sequence coding the Glutathione S-transferase (GST) gene (
The expression plasmid was introduced in E. coli lon−, ompT− BL21 and the E. coli trxB−/gor− deficient strain, Origami B. Monitoring of GST-huIFNα2b fusion protein expression was performed at 37° C. and at three IPTG concentrations (0.1, 0.5, 1 mM), and cell growth continued for 8 hours. The final OD at 600 nm was equal to 2 environ. The GST-hIFNα2b recombinant protein expression at 37° C. and at three different IPTG concentrations (0.1, 0.5, 1 mM) was analysed by 15% SDS-PAGE on both the supernatant and the cell pellet.
The expression profile of GST-hIFNα2b in E. coli BL21 strain is shown in
The presence of GST-huIFNα2b fusion protein in both the soluble and inclusion body fractions was confirmed by Western blot analysis using the anti-GST antibody (
Expression in E. coli Origami B strain of GST-hIFNα2b using the same plasmid gave a similar observation of higher ratio of insoluble recombinant protein at 37° C. (data not shown).
The effect of temperature and IPTG inducer concentration on the expression pattern of GST-hIFNα2b in E. coli strain BL21 was studied by western blot analysis. As shown in
Five thrombin concentrations, 10 U, 20 U, 50 U, 100 U, and 200 U, were used to cleave 100 μg of affinity-purified GST-IFNα2b fusion protein. The cleavage of the GST-hIFNα2b samples using the five thrombin concentration conditions was analysed on SDS-PAGE followed by a western blot analysis using anti-human IFNα polyclonal antibody. As shown in
To enhance the amount of the soluble GST-hIFNα2b protein expression and improve thrombin cleavage rate, we have engineered the sequence coding for the GST-IFN junction that includes the thrombin cutting site. As shown in
The cDNA sequence corresponding to the thrombin recognition site was analyzed using the <<E. coli Codon Usage Analysis 2.0>> software developed by Morris Maduro, which is available through the website http://www.lifesci.ucsb.edu/˜maduro/codonusage/usage2.0c.htm. The codon sequence analysis (
The engineered plasmid, called pGEX-Δ-hIFNα2b, was fully checked by DNA sequencing and used to transform E. coli BL21 and Origami strains.
To check the efficiency of thrombin cleavage of the engineered GST-IFN junction (sequence Δ), three thrombin concentrations (0 U, 20 U and 50 U) were used to cleave 100 μg of affinity-purified GST-Δ-hIFNα2b fusion protein produced by BL21 grown at 25° C., and Origami B grown at 37° C. The cleavage samples from the three thrombin concentration conditions were analysed on SDS-PAGE gels followed by a western blot analysis using anti-human IFNα polyclonal antibody (
The original and engineered plasmids were introduced in BL21 (lon−/ompT−) and Origami B (trxB−/gor−) E. coli strains, and the expression pattern of GST-hIFNα2b fusion protein was compared. Analysis was carried out by Western blot using the anti-GST antibody followed by ImageJ analysis. As shown in
To measure the amount of GST-rhIFNα2b fusion protein produced by Origami B, the recombinant protein was purified from the supernatant of a culture grown at 37° C. and 0.5 mM IPTG. Purification of GST-huIFNα2b was performed by affinity chromatography using a Glutathione Sepharose GSTrap column. The purification profile was analysed in a reduced, Coomassie stained SDS-PAGE gel (
The purified GST-hIFNα2b fusion protein was cleaved by thrombin protease to remove the GST moiety. Twenty units thrombin were used to digest 100 μg of GST-rhIFNα2b to completion (
The biological activity of the purified huIFNα2b preparation was determined by the antiviral and Gene Report assays (Meager, 2002). The antiviral assay is based on the ability of huIFNα2b to inhibit the cytopathic effect caused by encephalomyocarditis virus (EMCV) on the glioblastoma cell line 2D9. Furthermore, cell lines (HEK 293P) stably transfected with IFN-inducible promoter sequence (ISRE) linked to the SEAP gene (secreted alkaline phosphatase) were used to perform the Gene Report assay. The Relpot.xls Version 2.11 software [Scott Hutchinson/Amgen] was used to calculate the IFN biologic activity. The purified rhuIFNα2b from E. coli was calibrated against the IFNα WHO international standard (code: 95/566) (Meager, 2001) and exhibited a specific activity of 2×108 IU/mg in both assays.