PEDS Advance Access published online on April 12, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzm012
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A strategy for high-level expression of soluble and functional human interferon 
as a GST-fusion protein in E.coli
Molecular Biotechnology Group, Institute Pasteur, Tunis, Tunisia
1 To whom correspondence should be addressed. E-mail: dahmani.fathallah{at}pasteur.rns.tn; medfa{at}lycos.com
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Abstract |
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Escherichia coli is the most extensively used host for the production of recombinant proteins. However, most of the eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. To achieve high-level expression of soluble recombinant human interferon
(rhIFN) in E.coli, we have first constructed a recombinant expression plasmid (pGEX-hIFN2b), in which we merged the hIFN
2b cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac-inducible promoter. Using this plasmid, we have achieved 70% expression of soluble rhIFN2b as a GST fusion protein using E.coli BL21 strain, under optimized environmental factors such as culture growth temperature and inducer (IPTG) concentration. However, release of the IFN moiety from the fusion protein by thrombin digestion was not optimal. Therefore, we have engineered the expression cassette to optimize the amino acid sequence at the GST–IFN junction and to introduce E.coli preferred codon within the thrombin cleavage site. We have used the engineered plasmid (pGEX-
-hIFN2b) and the modified E.coli trxB–/gor– (Origami) strain to overcome the problem of removing the GST moiety while expressing soluble rhIFN2b. Our results show the production of soluble and functional rhIFN2b at a yield of 100 mg/l, without optimization of any step of the process. The specific biological activity of the purified soluble rhIFN2b was equal to 2.0 x 108 IU/mg when compared with the WHO IFN standard. Our data are the first to show that high yield production of soluble and functional rhIFN2b tagged with GST can be achieved in E.coli.
Keywords: E.coli/expression/interferon/recombinant/soluble
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionAcknowledgementReferences |
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Interferons (IFNs) are a group of naturally produced glycoproteins endowed with anti-viral, anti-proliferative and immuno-modulatory properties (Pfeffer, 1997
; Pestka et al., 1987) as well as an analgesic action (Wang et al., 2000, 2002). The medical potential of IFNs was soon recognized, as demonstrated by the approval in 1986 of recombinant hIFN2a (rhIFN2a) (Roferon-A), and later IFN2b (Intron-A), as drugs for the treatment of malignant and viral diseases (Gutterman, 1994; Lauer, 2001; Mahon et al., 2002; Motzer et al., 2002). Most of the marketed pharmaceutical grade recombinant IFN has since been produced and purified from Escherichia coli.
The E.coli recombinant protein expression system has been, and still is, the system of choice for the production of IFN. Indeed, IFN 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 IFN cDNA was achieved directly in E.coli soon after it was first cloned (Goeddel et al., 1980; Nagata et al., 1980; Pestka, 1983; Mizoguchi et al., 1985; Pestka et al., 1987; Barron and Narula, 1990).
Several promoter systems were chosen to achieve high intracellular expression levels (Laplace et al., 1988; Boyer et al., 1992; Swaminathan and Khanna, 1999; Babu et al., 2000; Lim et al., 2000; Bedarrain et al., 2001; Neves et al., 2004; Srivasta et al., 2005).
However, IFN protein expressed in large amount in E.coli often precipitates into insoluble aggregates called inclusion bodies (Swaminathan and Khanna, 1999; Bedarrain et al., 2001; Srivasta et al., 2005) that are, in general, misfolded proteins and thus biologically inactive (Villaverde 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. The purification of soluble recombinant proteins is more cost-effective and less time-consuming than refolding and purification 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 favorable 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 toward creating inclusion bodies (Lilie et al., 1998). In several instances, the use of an affinity tag such as glutathione S-transferase (GST) for recombinant protein purification has proven to be effective in improving the recombinant protein solubility (Smith and Johnson, 1988). Other affinity tags have also been reported to improve protein yield, to prevent proteolysis and to increase solubility in vivo (Makrides, 1996; Sorensen and Mortensen, 2005a,b).
Several other expression systems were used to overcome the problem of inclusion bodies and improve protein solubility for the expression of rhIFN. 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 rhIFN; however, none of them allowed the yields obtained in E.coli within inclusion bodies.
Although the production of recombinant proteins in E.coli is well established, there are numerous factors that may present obstacles for successful production and purification of soluble recombinant proteins (Baneyx and Mujacic, 2004). Two main approaches are generally used, separately or in combination, to favor expression of soluble recombinant proteins. The first one calls for optimization of environmental factors such as growth temperature, media and concentration of gene expression inducers (Baneyx and Mujacic, 2004), and the second one involves genetic engineering of the target protein (Makrides, 1996; Sorensen and Mortensen, 2005a,b).
In this paper, we report a strategy that combines both approaches to show that production of soluble rhIFN can be achieved in E.coli. We first describe the investigation of the effect of environmental factors such as growth temperature and the concentration of the transcription inducer (IPTG) in improving the solubility of the fusion protein GST-IFN2b produced in E.coli. Then, we describe 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, which allowed the production of 100 mg/l of pure, soluble and functional rhIFN2b.
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Methods |
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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, Wisconsin, USA), and the E.coli lon– and ompT– BL21 protease-deficient strains (Amersham Pharmacia Biotech) were used as host strains for recombinant GST--hIFN2b expression.
Construction of recombinant pGEX-hIFN2b expression vector
hINF2b 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 TRIZOLTM mRNA extraction method as described by the manufacturer (Invitrogen). The cDNA corresponding to the IFN2b published sequence (Pestka, 1983) was amplified using a forward primer that introduced an EcoRI site at the 5' end of the gene (5'-TGGAATTCTGTGATCTGCCTC AAACCCA-3') and a reverse primer containing the XhoI site at the 3' end of the gene (5'-CGCTCGAGTCATTCCTTACT TCTTAAACTTTC-3'). The purified PCR product was digested with EcoRI and XhoI restriction enzymes and inserted into the plasmid pGEX4T1 (Amersham Biosciences) to generate the pGEX-hIFN2b expression vector. Screening of pGEX4T1/IFN2b recombinant plasmids containing the cDNA sequence encoding hIFN2b 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--hIFN2b expression vector
The pGEX-hIFN2b 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 Fig. 1: F (TGT GAT CTG CCT CAA ACC CAC) and R (GGA GCC ACG CGG AAC CAG). Finally, screening of pGEX--hIFN2b mutant clones was performed by restriction analysis using EcoRI restriction enzyme as recommended by the manufacturer (Amersham Biosciences), and by DNA sequencing analysis as described earlier.
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Analytical expression of recombinant GST-rhIFN
2b
The Origami B and BL21 E.coli cell lines were transformed with the wild-type pGEX-rhIFN2b plasmid and the GST–IFN junction re-engineered pGEX--hIFN2b plasmid, using the TSS method following standard protocols.
Starter cultures of 5 ml Luria–Bertani (LB) 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. One milliliter 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 (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-hIFN2b wild-type and GST--hIFN2b 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 of 2 at 600 nm.
Extraction of GST-hIFN2b recombinant protein
Cells from induced and un-induced cultures were harvested by centrifugation (4000g, 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 4000g for 30 min, and finally stored at –70°C until use. Protein extraction was performed by resuspending the cell pellet in one-fifth 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-s sonication steps. The supernatant was collected by centrifugation at 4°C for 30 min at 13 500 rpm and stored for GST--hIFN2b 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--hIFN2b 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-hINF 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--hIFN2b in Origami B lysate versus rhIFN2b obtained after thrombin cleavage and the two-step purification was also determined by a quantitative in-house developed ELISA assay. Dilution series containing 0–570 pg of HPLC-purified soluble IFN2b produced in our laboratory were included in each assay to construct a standard curve. Recombinant proteins were detected using anti-hINF biotin-labeled monoclonal antibody (ENDOGEN Searchlight) and a colorimetric detection system using a streptavidin–horseradish peroxidase (HRP) conjugate (Amersham Biosciences).
The purity of the recombinant hIFN2b 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-hIFN2b recombinant protein
The supernatant containing the soluble GST-hIFN2b 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 an OD of 280 nm returned to baseline. Once the baseline was stable, elution of the bound GST--hIFN2b recombinant protein was carried out using six 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-hIFN2b 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 earlier.
Twenty units of thrombin solution were added to 100 µg of eluted fusion protein and incubated at room temperature (+22°C) for 20 h.
Size-exclusion purification step of hIFN2b 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 rhIFN2b peak was eluted. The chromatographic profile was evaluated by Coomassie Blue and silver-stained SDS–PAGE gels.
Biological activity of rhIFN2b
The biological activity of the recombinant hIFN2b preparation was determined by the anti-viral and Gene Report assays as described by 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 rhIFN2b required to produce anti-viral activity equivalent to that expressed by 1 IU hIFN2b reference standard (code: 95/566; Division of Immunobiology; National Institute for Biological Standards and Control, Potters Bar, UK).
Clone stability was checked after 6 months of continuous culture by plasmid DNA preparation and DNA sequencing of the expression cassette.
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Results |
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Cloning of the hIFN
2b cDNA
hINF2b 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 GST gene (Fig. 1) and under the control of the IPTG-inducible tac promoter. The nucleotide sequence of the selected clone was checked by automated DNA sequencing analysis using 5' pGEX sequencing primer.
Expression of GST-hIFN2b fusion protein
The expression plasmid was introduced in E.coli lon–, ompT– BL21 and the E.coli trxB–/gor– deficient strain, Origami B. Monitoring of GST-hIFN2b fusion protein expression was performed at 37°C and at three IPTG concentrations (0.1, 0.5 and 1 mM), and cell growth continued for 8 h. The final OD at 600 nm was equal to two environ. The GST-hIFN2b recombinant protein expression at 37°C and at three different IPTG concentrations (0.1, 0.5 and 1 mM) was analyzed by 15% SDS–PAGE on both the supernatant and the cell pellet.
The expression profile of GST-hIFN2b in E.coli BL21 strain is shown in Fig. 2. The full fusion protein was present in both soluble (Fig. 2A) and inclusion bodies fractions (Fig. 2B). Furthermore, a band of 26 kDa corresponding to free GST was constantly observed. The expression of GST-hIFN2b recombinant protein expression as inclusion bodies was highest at 37°C and induction using a concentration of 1 mM IPTG. Therefore, an IPTG concentration of 0.1 mM was retained to compare the level of GST-hIFN2b expression at 37°C in both supernatant and inclusion body fractions by western blot analysis.
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The presence of GST-hIFN
2b fusion protein in both the soluble and inclusion body fractions was confirmed by western blot analysis using the anti-GST antibody (Fig. 3A). However, the signal from the soluble fraction was much weaker as assessed by analysis of the western blot film using the ImageJ software. We observed an expression ratio of 67.39% insoluble (present in inclusion bodies) and over 32.61% soluble (present in the soluble fraction) GST-hIFN2b fusion protein (Fig. 3B).
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Expression in E.coli Origami B strain of GST-hIFN
2b using the same plasmid gave a similar observation of higher ratio of insoluble recombinant protein at 37°C (data not shown).
Enhancement of the level of soluble GST-hIFN2b expressed in BL21 strain at reduced temperature
The effect of temperature and IPTG inducer concentration on the expression pattern of GST-hIFN2b in E.coli strain BL21 was studied by western blot analysis. As shown in Fig. 4A, the level of soluble GST-hIFN2b expression increased when cell growth was carried out at 25°C and using 0.1 or 0.5 mM IPTG for induction, as compared with growth at 37°C. These observations were confirmed by analysis of the western blot films using the ImageJ software. The results shown in Fig. 4B and C show that the amount of soluble intracellular GST-hIFN was increased more than 2-fold when culturing was performed at lower growth temperature (i.e. 25°C) and induction using either 0.1 or 0.5 mM IPTG. Furthermore, the level of soluble GST-hIFN2b dropped to 34.68% when induction was carried out using 1 mM IPTG, even at 25°C growth temperature (Fig. 4D).
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Incomplete thrombin cleavage of GST-IFN
2b fusion protein
Five thrombin concentrations, 10, 20, 50, 100 and 200 U, were used to cleave 100 µg of affinity-purified GST-IFN2b fusion protein. The cleavage of the GST-hIFN2b samples using the five thrombin concentration conditions was analyzed on SDS–PAGE followed by a western blot analysis using anti-hINF polyclonal antibody. As shown in Fig. 5, the cleavage rate was not complete and did not significantly increase when thrombin concentration was increased. Identical results of incomplete cleavage were observed using different sources of thrombin (i.e. different manufacturers).
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Engineering of the GST-hIFN
2b expression cassette
To enhance the amount of the soluble GST-hIFN2b 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 Fig. 1, this engineering consisted of deleting the three extra residues (Pro, Glu and Phe) at positions –1, –2 and –3 from the first amino-terminal residue of hIFN, and on optimizing the codon corresponding to the glycine at position –5.
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 (Fig. 6A and B) shows the presence of a rare glycine codon, GGA (–5), classified as the 11th rarest codon in E.coli mRNA, occurring at a frequency of 0.8% (Wada et al., 1992). Site-directed mutagenesis (Rabhi et al., 2004) was used to delete this codon from the GST-INF2b linker sequence, the Phe (at position –1) and Glu (at position –2) introduced in the original vector by the EcoRI cloning site, and the rigid Pro (at position –3) amino acid residues. Furthermore, the rare glycine codon, originally GGA (–5), has been replaced by GGC according to the optimal codon usage for E.coli.
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The engineered plasmid, called pGEX-
-hIFN2b, was fully checked by DNA sequencing and used to transform E.coli BL21 and Origami strains. Improvement of thrombin cleavage rate
To check the efficiency of thrombin cleavage of the engineered GST–IFN junction (sequence ), three thrombin concentrations (0, 20 and 50 U) were used to cleave 100 µg of affinity-purified GST--hIFN2b 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 analyzed on SDS–PAGE gels followed by a western blot analysis using anti-hINF polyclonal antibody (Fig. 7). As shown in Fig. 7, the cleavage of rhIFN2b from the GST partner was optimal using 50 U of thrombin. This result shows that thrombin cleavage is improved when the junction of GST-hIFN2b is engineered.
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Enhancement of the level of soluble rGST-hIFN
2b using the engineered clone
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-hIFN2b fusion protein was compared. Analysis was carried out by western blot using the anti-GST antibody followed by ImageJ analysis. As shown in Fig. 8, the amount of soluble GST-hIFN2b protein was increased more than 2-fold when expression is carried out at 25°C and 0.5 mM IPTG using the Origami B host strain. Analysis of the expression of soluble GST-hIFN2b fusion protein by the Origami B strain bearing the GST–IFN expression plasmid with the engineered junction, cultured at either 37°C (Fig. 9A) or 25°C (Fig. 9B), shows that over 80% of the protein was in the soluble fraction. It is interesting to note that the Origami B host strain is able to efficiently process the expression of soluble GST-hIFN2b fusion protein at 37°C. Furthermore, when using the plasmid with the original GST–IFN junction sequence, the expression of soluble GST-hIFN2b in Origami B could not reach more than 50% even when grown at the optimal conditions defined for Origami B (i.e. growth at 37°C and induction using 0.5 mM IPTG) (Fig. 9C). This result shows that the expression of soluble form of recombinant GST-hIFN2b depends on culture conditions (i.e. temperature, IPTG inducer concentration) and also on the host strain used for expression.
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Purification of the soluble fraction of hIFN
2b expressed in E.coli
To measure the amount of GST-rhIFN2b 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-hIFN2b was performed by affinity chromatography using a Glutathione Sepharose GSTrap column. The purification profile was analyzed in a reduced, Coomassie-stained SDS–PAGE gel (Fig. 10). An yield of 1.22 g/l of purified GST-hIFN2b was obtained.
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The purified GST-hIFN
2b fusion protein was cleaved by thrombin protease to remove the GST moiety. Twenty units of thrombin were used to digest 100 µg of GST-rhIFN2b to completion (Fig. 11, lane T). The 26-kDa GST moiety and the 37-kDa thrombin protease were removed by size-exclusion chromatography. A final yield of 100 mg of pure rhIFN2b was obtained. Purity was checked by Coomassie-stained SDS–PAGE gel (Fig. 11).
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Biological activity of soluble hIFN
2b expressed in E.coli
The biological activity of the purified hIFN2b preparation was determined by the anti-viral and Gene Report assays (Meager, 2002). The anti-viral assay is based on the ability of hIFN2b to inhibit the cytopathic effect caused by encephalomyocarditis virus on the glioblastoma cell line 2D9. Furthermore, cell lines (HEK 293P) stably transfected with IFN-inducible promoter sequence (ISRE) linked to the secreted alkaline phosphatase (SEAP) gene were used to perform the Gene Report assay. The Relpot.xls Version 2.11 software [Scott Hutchinson/Amgen] was used to calculate the biological activity of IFN. The purified rhIFN2b from E.coli was calibrated against the INF WHO international standard (code: 95/566) (Meager et al., 2001) and exhibited a specific activity of 2 x 108 IU/mg in both assays.
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Discussion |
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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. We have developed a strategy to drive the expression of hIFN
2b in E.coli from aggregated protein in the inclusion bodies to soluble and properly folded cytoplasmic protein. A well-known approach to limit the in vivo aggregation of recombinant proteins consists of cultivation at reduced growth temperature. Lowering the concentration of the inducer, in expression systems using an inducible promoter, also contributes to enhancing the solubility of recombinant protein produced in E.coli (Schein, 1989). This strategy has proven to be effective in improving the solubility of a number of difficult-to-express proteins (Vasina and Baneyx, 1997). The aggregation reaction is favored at high temperature because of the strong temperature dependence of hydrophobic interactions (Kiefhaber et al., 1991). It was reported that the direct consequence of decreasing temperature is the partial elimination of heat-shock proteases that are induced during foreign protein overexpression (Chesshyre and Hipkiss, 1989). Moreover, expression and activity of a number of E.coli chaperones are increased at low temperature around 30°C (Mogk et al., 2002; Ferrer et al., 2003). Thus, the increased stability and potential correct folding at low temperature are partially explained by these factors. Furthermore, it was reported that low induction levels have been found to increase soluble protein expression (Weickert et al., 1996; Baneyx and Mujacic, 2004).
Our strategy was designed for expression of hIFN2b as a fusion protein with GST under the control of the tac promoter. We have used the approach described above as a first step toward expressing soluble IFN2b in E.coli. Our data show that we have achieved enhancement (up to 70%) of the expression of IFN2b as an intracellular soluble fusion protein in the E.coli BL21 strain. This result was achieved at a growth temperature of 25°C and induction with 0.1–0.5 mM IPTG. However, this soluble GST-hIFN2b fusion protein could not be digested efficiently with thrombin to release the IFN moiety. Furthermore, we have consistently observed the presence of free GST along with the fusion protein. The possible presence of two types of clones was ruled out. We have concluded at this stage that the inefficient thrombin digestion might be due to a sub-optimal folding of the fusion protein at the junction of GST and IFN. Furthermore, we suspected translational pausing as being the underlying cause for the presence of free GST. Possible reasons for translational pausing during protein expression in E.coli host strains were reviewed in the literature (Tsalkova et al., 1999). It has been demonstrated that a strong correlation exists between the frequency of codon usage and the level of its cognate tRNA (Ikemura, 1981). We have analyzed the nucleotide and amino acid sequence at the junction between GST and IFN to design the second step of our strategy, which consists of engineering the sequence at this junction area to improve the thrombin cleavage and eventually prevent free GST expression.
Amino acid sequence analysis of the junction area revealed that the Pro at position –3 may influence the linker architecture, because its ring structure makes it more conformationally restricted and minimizes the flexibility of the thrombin recognition site. Moreover, the presence of a second Pro in the thrombin linker peptide sequence may form an X-Pro linkage, which is the most common cis peptide bond. This bond is known to show less preference for the trans configuration, because the nitrogen of proline is bound to two tetrahedral carbon atoms, limiting the steric differences between the trans and cis form and resulting in a potential steric clash between the two adjacent GST and hIFN2b protein units. Moreover, because of the presence of the proline pyrrolidine ring, the 
angle of rotation is blocked at approximately –65°, resulting in a restricted set of allowed 
angles, which may cause a freedom of rotation limit about the bond between the nitrogen and -carbon. This may forbid the fusion protein to fold in many different ways. Furthermore, the steric clash occurring between the GST and hIFN2b protein units may decrease the accessibility of thrombin protease toward its specific recognition site.
Codon preference analysis at the junction area showed the presence of the rare glycine codon GGA (0.8%). Engineering was carried out to delete the Pro–Glu–Phe introduced by the EcoRI site to enhance the flexibility, and thus the accessibility of thrombin to the cleavage recognition site, and reduce the potentially immunogenic residues left after thrombin cleavage at the IFN2b amino terminus. Changing the glycine codon GGA to GGC was carried out to prevent potential translational pausing.
We have also considered the use of a different E.coli strain, E.coli trxB–/gor– Origami B. The choice of E.coli trxB–/gor– Origami B as an alternative host was dictated by the fact that proper folding of the IFN molecule necessitates the formation of two disulfide bridges between Cys 1–Cys 98 and Cys 29–Cys 138. Indeed, in the cytoplasm of normal E.coli strains, cysteines are actively kept in the reduced state by a pathway involving thioredoxin reductase and glutaredoxin (Ritz et al., 2001). Disruption of the trxB and gor genes, encoding the two major reductases of E.coli, allows formation of disulfide bonds in the E.coli cytoplasm (Xiong et al., 2005). The E.coli trxB–/gor– Origami B strain has been selected in several expression situations where formation of disulfide bonds was successfully achieved (Bessette et al., 1999; Lauber et al., 2001; Lobel et al., 2002; Venturi et al., 2002). Interestingly, close to 100% of GST-IFN2b expressed by the Origami B from our engineered clone was soluble when culture was carried out at 37°C. This result shows that the E.coli trxB–/gor– Origami B strain is a good host for high expression level, soluble and functional hIFN2b from our engineered GST-IFN2b clone. Cultivation of this clone at high cell density at 37°C will probably allow the attainment of production level higher than the 300 mg/l of INF that Babu et al. (2000) obtained from inclusion bodies by optimized E.coli cultivation at high cell density.
In conclusion, we have designed and performed a strategy that allowed us to develop a genetically engineered E.coli clone able to produce a soluble and active hIFN2b, using the double mutant trxB–/gor– E.coli Origami B cell line grown at 37°C. However, the 26 kDa free GST co-expressed in E.coli has to be further investigated with studies on molecular aspects such as mRNA stability, folding and rare codon usage in the E.coli host expression system.
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Footnotes |
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Edited by Fabrizio Chiti
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Acknowledgement |
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Dr Anthony Meager, Immunobiology Division, National Institute for Biological Standards and Control, Potters Bar, UK, for his help in carrying out the biological activity of the rhIFN
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References |
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Received September 9, 2006; revised December 23, 2006; accepted January 3, 2007.
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