High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
© Lee et al; licensee BioMed Central Ltd. 2011
Received: 6 April 2011
Accepted: 23 July 2011
Published: 23 July 2011
Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention.
Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay.
Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.
Chicken anemia virus (CAV), is the causative agent of chicken anemia disease. The disease results in severe anemia, lymph organ atrophy and immunosuppression [1–3]. CAV is the sole member of the genus Gyrovirus of the Circoviridae family. Histopathological studies have also shown that CAV infection leads to aplasia of the bone marrow and the destruction of T lymphoid tissue in young chickens [4, 5]. When chicks are infected with CAV, the mortality rate is often as high as 55% and morbidity rates of 80% have been reported . Therefore, worldwide, CAV is an economically important veterinary virus that can seriously affect the poultry industry.
VP1 protein (51 kDa) is the sole structural protein found within the CAV capsid. At a very late stage of the virus life cycle, the assembled virus particles created by VP1 protein spread into various other tissues and organs in the chicken, such as the thymus, spleen and liver . Immunogenicity studies have shown that VP1 allows the elicitation of host-produced virus neutralizing antibodies . Thus, VP1 is thought to be a good candidate for use as an immunogen when developing subunit vaccines and diagnostic kits [7, 8]. Up to the present, a number of different expression systems have been used to produce VP1 protein, including E. coli, baculovirus-insect cells and plant cells [9–12]. However, production of the recombinant full-length VP1 protein has generally been unsuccessful because of a failure to express a span of amino acids at the N-terminus of the VP1 protein that is highly rich in arginine residues [9, 11]. Furthermore, VP1 has been proposed to be cytotoxic in an E. coli expression system . Once the N-terminus of VP1 is deleted, protein expression is improved significantly . Nevertheless, the N-terminus of VP1 may still be involved in eliciting neutralizing antibodies because it contains some functional epitopes. Thus, there is a need to overcome the difficulties that have been encountered during production of full-length VP1 protein using an E. coli expression system. If successful, it would not only allow the efficient development of a subunit vaccine that is active against chicken anemia virus infection but the recombinant protein will also be potentially useful when developing diagnostic kits for the clinical detection of CAV infection.
As can be seen from the above information, E. coli has a number of disadvantages and limitations when producing CAV VP1 protein. However, the expression of VP1 protein in E. coli is still an attractive alternative to the current production system when assessed with respect to cost, time and operational considerations. In this study, we investigated the effect on expression of VP1 of changes in codon usage for various amino acids within the N-terminus of VP1 gene; this is because these amino acids are encoded by codons that are rarely used by E. coli. The codons within the N-terminus of the VP1 protein were optimized using prediction software and changed to the preferred codon usage for E. coli. In addition, VP1 was cloned into the expression vector, pGEX-4T-1, in order to create a glutathione-S-transferase (GST) fusion protein with the N-terminus of VP1. Expression of the modified VP1 genes was examined in various E. coli strains and changes in CAV VP1 protein expression determined. To the best of our knowledge, the yield of E. coli expressed recombinant VP1 in this study, after codon optimization of the N-terminal region, is the highest known to date.
GST fusion tag enhances the expression of recombinant CAV VP1 protein in E. coli
Growth profile and expression levels of recombinant GST-VP1 protein in various different E. coli strains
Optimization of codon usage enhances the expression of recombinant CAV VP1 protein in E. coli
Purification of recombinant GST-opt-VP1 protein using GST affinity chromatography
Application of recombinant CAV VP1 protein to an indirect ELISA
In this study, the specific aim was to establish a prokaryotic expression system for the production of full-length recombinant CAV VP1 protein. A prokaryotic expression system has several advantages when expressing a heterologous recombinant protein; these include cost-effectiveness, time-saving, ease of production and the fact that the antigenic characterization of recombinant protein are easier to confirm. Therefore, E. coli expression systems are the most used approaches when screening for the expression of a foreign protein . Indeed, among above points are many of the critical factors associated with choosing a suitable production system for the development of a subunit vaccine and diagnostic kit. However, in previous studies, there have many difficulties when expressing full-length CAV VP1 protein using E. coli as the expression host [9, 11]. In addition to VP1 protein expression, proteolysis in E. coli also seems to occur resulting in the degradation of the VP1 protein . Although an N-terminus deleted VP1 has been successfully expressed in E. coli, nevertheless, the use of a truncated form of the CAV VP1 protein might hampers its usefulness in serological tests or as a subunit vaccine. Thus, the problems caused by the lack of a truly effective system for producing VP1 protein still needed to be overcome.
Using Genscript OptimumGene™ bioinformatic software, the rare codons in E. coli that exist in the wild-type CAV VP1 gene were analyzed. These amino acid residues include arginine, leucine, proline and lysine, which are common in the N-terminus region of the VP1 protein (1-321 bp). Rosenberg et al proposed that the abundance of rare codon near the 5'-end of the gene might affect the efficiency of protein translation and this suggested the present approach to increasing expression of the full-length CAV protein .
Both of porcine circovirus (PCV) and CAV are members of the circovirus family. PCV also has similar basic amino acid residues rich in rare codons at the N-terminus of the capsid protein and this also results in decreased protein expression . However, Liu et al successfully overcame this problem by fusing a maltose-binding protein (MBP)-His × 8 tag to the PCV capsid protein for expressing and the resulting fusion protein in E. coli. Why the presence of a MBP-His × 8 tag improves protein expression remains unclear, but one possibility is improved protein solubility . Similarly, in the present study, the addition of a GST tag to the VP1 protein improved expression in E. coli significantly compared to a His × 6 tag (Figure 2B). Thus it would seem that some fusion tags are able to promote better expression in E. coli than other tags, perhaps by aiding the correct folding of their fused partner protein . This report is the first to show that full-length recombinant CAV VP1 protein can be expressed successfully in E. coli by adding a GST tag.
Presently, it had been recognized that VP1 is a sole structural protein. This protein is not only an immunogen for used as vaccine candidate but also suitable applied as a coating antigen of ELISA for diagnostic purpose [7, 8, 10]. However, there has no any prokaryotic expression system which successful employed for production of full-length VP1 of CAV. In addition, the full-length VP1 of CAV involved all of the epitopes which can elicit virus neutralizing antibodies of the host. For this study, the full-length VP1 protein was soluble with 80% of total expressed protein, and retained the antigenic characteristics of the native VP1 (Figure 7 and 9B). Thus, it implied that the most important immunodomain, the VP1 of the CAV capsid, can highly yield produced herein, and will be used conveniently for developing recombinant vaccine in the other further studies.
Although the full-length of recombinant CAV VP1 protein was able to be expressed in E. coli by fusing the VP1 protein with a GST tag, the yield of GST-VP1 remained relatively low at (approximately 3.78 mg/ml (Figure 4B). This may be a result of poor protein stability, the toxicity of CAV VP1 protein or others factor including perhaps rare codons within the VP1 gene [9, 11]. When the full-length VP1 protein was expressed in BL21 (DE3), BL21 (DE3)-pLysS and BL21 (DE3)-CodonPlus-RP, the highest level of the full-length VP1 protein occurred with in BL21 (DE3)-pLysS. This perhaps indicates that stain BL21 (DE3)-pLysS might be more tolerant of any cytotoxicity. BL21 (DE3)-CodonPlus-RP was able to reach an expression level of 1.71 mg/ml at 1 h post-induction, which is about 30% higher than in BL21 (DE3) and BL21 (DE3)-pLysS under the same conditions (Figure 4B, C and 4D). One reason for the increase in full-length VP1 expression by BL21 (DE3)-CodonPlus-RP cells might be the presence of extra copies of the tRNA argU, proL genes. However, such over-expression might exhaust endogenous energy supplies, which is deleterious to the cells, especially when they are induced with IPTG induction. In this context, it was found that IPTG induction of over 2 h, when the bacterial cells were grown at 37°C, failed to increase the yield of VP1 further to any significant degree. Also noteworthy is the fact that the growth profile of BL21 (DE3)-CodonPlus-RP during the course of expression of GST-VP1 was not changed (Figure 4A). This phenomenon might cause by a balancing of the steady-state growth condition of the strains with yield, which suggests that the strain had reached its maximum possible growth rate for expressing full-length CAB VP1 .
In such circumstances, it seems likely that optimization of codon bias would be a useful strategy to improve the expression of full-length CAV VP1 protein. By engineering the foreign gene to use E. coli's preferentially used codons; this will improve translation efficiency without the need to supply extra copies of the rare tRNA genes [21–23]. When the rare codons within the N-terminus of the VP1 gene were optimized, expression of full-length optVP1 protein in BL21 (DE3)-pLysS significantly increased (Figure 5B, 6B). The comparably higher yield of optVP1 to wild type VP1 or any other published expression system after the rare codons had been optimized within the N-terminus of VP1 protein and using BL21 (DE3)-pLysS, was approximately 4.6 fold greater than for wild type VP1 at 1 h post-induction. Notably, an increase in induction to greater than 2 hours was not further increased the accumulation of optVP1 protein. The limit may be the protein burden within the E. coli cells, which eventually leads to growth arrest (Figure 6A, B). However, using a fed-batch based cultivation system, this difficulty might be overcome and allow even higher yields of full-length VP1 protein to be obtained in the future.
The expression of recombinant full-length VP1 protein of CAV using a prokaryotic system was established successfully and the yield was greatly improved by making various adjustments to the expression system. Optimization of codon usage within N-terminal region of VP1 protein was a most convenient and cost-effective way of increasing the expression of full-length CAV VP1 protein in E. coli. This paves the way for large-scale production of the full-length VP1 protein using this approach, which will allow the full-length VP1 to be used for the development of a subunit vaccine or a diagnostic test.
Bacterial strain and cells inoculation
BL21 (DE3) was obtained from Invitrogen Life Technologies (Carlsbad, CA). BL21-CodonPlus(DE3)-RP and BL21(DE3)-pLysS were purchased from Stratagene (La Jolla, CA). Overnight cultures (10 ml LB medium in 50 ml flasks) were grown at 37°C for strain activation. Then 0.5 ml of the overnight culture were inoculated into 50 ml LB medium and grown at 37°C for around 3 h by which time the optical density of culture had reach 0.5 of OD600 for cell transformation or protein expression.
A 1350 bp of cDNA encoding the full-length CAV VP1 protein was cloned into pET28a (Novagen, Madison, WI) earlier (Figure 1A, panel a) (Lee et al., 2009) . Using a specifically designed primer set, forward primer CAV VP1 F: 5'-GCGGAATTCATGGCAAGACGAGCTCGCAGA-3' and reverse primer CAV VP1-R: 5'-GGGCTCGAGTCAGGGCTGCGTCCCCCAGTA-3', respectively containing the EcoR I and Xho I sites, the full-length VP1 gene was amplified using the plasmid pET28a-VP1 as template DNA, and then cloned into the plasmid pGEX-4T-1 (GE healthcare, Piscataway, NJ), which was then designated to be pGEX-4T-1-VP1 (Figure 1A, panel b). This construct carries the VP1 gene flanked by the N-terminal glutathione-S-transferase (GST) tag and will express a GST-VP1 fusion protein. To generate the plasmid harboring the codon optimized nucleotide sequence, a codon optimized fragment encoding the first 107 amino acid residues of the N-terminus of the VP1 capsid protein was fused with the C-terminus of the VP1 protein using the 5' and 3' ends of above two DNA fragments and overlapping PCR . The PCR used three different primer sets, Opt-F1 (5'-CCCGAATTCATGGCTCGTCGTGCTCGTCGT-3') and Opt-R1 (5'-CGCTAGCAGGAACTCTTTCAGGTT-3'), Opt-F2 (5'-AACCTGAAAGAGTTCCTGCTAGCG-3') and CAV VP1-R for the first amplification and then CAV VP1 F and VP1-R for the second amplification. The result was a fragment containing the entire cDNA, Opt-VP1, which was then ligated into pGEX-4T-1 and designated to be pGEX-4T-1-Opt-VP1 (Figure 1A, panel c). The constructed recombinant plasmids were transformed into One Shot® Top10 (Invitrogen, CA) chemically competent E. coli for the maintenance of the recombinant plasmids and protein expression. Transformants that contained a gene of the correct size by PCR were then checked using restriction enzyme digestion and DNA sequence analysis.
Codon optimization of cDNA of the N-terminus VP1 protein
The codons of the cDNA of the N-terminus of VP1 protein (321 bp) were optimized based on the codon preference of E. coli using Genscript OptimumGene™ designing software. The codon-optimized cDNA of the N-terminus of VP1 protein was synthesized by Genomics Biosci & Tech Co. (Taipei, Taiwan) and then assembled into the C-terminus of VP1 protein to give the entire VP1 gene using the approach described above .
Expression of full-length VP1 protein after codon optimized in recombinant E. coli
The host E. coli strains BL-21(DE3), BL21-CodonPlus(DE3)-RP and BL21 (DE3)-pLysS have different recombinant constructions and these strains were used for protein induction and expression. The recombinant strains were grown in LB medium in the presence of kanamycin (50 μg/ml), ampicillin (50 μg/ml) or chloramphenicol (34 μg/ml) as appropriate at 37°C. When the culture had reached an optical density (OD600) of 0.5, isopropyl-β-D-thiogalactopyronoside (IPTG) at different concentrations was added to induce protein expression and then growth was continued for 4 h. After IPTG induction, samples of the cells were harvested using centrifugation for 10 min at 10,000 rpm. To determine the protein expression, the cell pellets were resuspended in 1xSDS-PAGE sample buffer and boiled for 5 min. The resulting lysates were then Western blotted after separation on a 12.5% SDS-PAGE. The expression level and concentration of the recombinant VP1 proteins were determined as described in a previous study .
Purification of recombinant VP1 protein using GST affinity chromatography
To purify the recombinant VP1 proteins, the cells were spun down from 50 mL of culture supernatant and resuspended in GST resin binding buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3). They were sonicated on ice three times for 3 minutes with a 20% pulsed activity cycle (MISONIX Sonicator® 300). The lysate was then centrifuged for 10 min at 10,000 rpm to remove the cell debris. The resulting cell supernatant was injected into a GSTrap FF affinity column (GE healthcare, Piscataway, NJ) with 1 mL of GSTrap resin at a flow rate of 0.5 ml/min. The packed column was washed with a 10-fold volume of binding buffer, and then the bound proteins were eluted with elution buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0) at 1 ml/min. Each fraction consisted of 1 ml of elutant. The fractions were monitored at OD280 using the optical unit of a liquid chromatography system (AKTAprime plus, GE Healthcare BioScience AB, Uppsala, Sweden). A putative peak containing the recombinant CAV viral protein was identified and the eluant collected for analysis. The total protein concentration of each fraction was determined using a Micro BCA kit (Pierce, Rockford, IL) using bovine serum albumin as the reference protein. The purity of the protein sample was analyzed using aliquots of the fractions by 12.5% SDS-PAGE and Western-blotting with appropriate antibodies.
To confirm the identity of the recombinant GST-opt-VP1 protein directly expressed in E. coli or purified by GSTrap FF column, the proteins were separated by 12.5% SDS-PAGE. The relevant band was then cut out from the 12.5% SDS-PAGE gel after Coomassie blue staining and digested with trypsin. The resulting samples were subjected to the MALDI-TOF-MS mass spectrometry (ESI-QUAD-TOF) to allow amino acid sequence identification of the protein, as described in a previous study .
The antigenicity of E. coli expressed VP1 protein after purification was evaluated by ELISA assay. Briefly, 2 μg per well of diluted VP1 protein was coated onto a 96 wells plate for 1 h at 37°C. After washing, CAV-infected positive serum was added and reacted for one another hour at 37°C. Subsequently, following washing, secondary antibodies were added and this was followed by color development as described in a previous study .
This work was supported by the grant from China Medical University of Taiwan (CMU97-284 & CMU97-191) and the National Science Council (NSC 95-2313-B-039-004-, NSC96-2313-B-276-001-MY3), Taiwan.
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