High yield production of pigeon circovirus capsid protein in the E. coliby evaluating the key parameters needed for protein expression

Abstract

BackgroundPigeon circovirus (PiCV) is considered to be a viral agent central to the development of young pigeon disease syndrome (YPDS). The Cap protein, a structural protein encoded by the cap (or C1) gene of PiCV, has been shown to be responsible for not only capsid assembly, but also has been used as antigen for detecting antibody when the host is infected with PiCV. The antigenic characteristics of the Cap protein potentially may allow the development of a detection kit that could be applied to control PiCV infection. However, poor expression and poor protein solubility have hampered the production of recombinant Cap protein in the bacteria. This study was undertaken to develop the optimal expression of recombinant full-length Cap protein of PiCV using an E. coli expression system.ResultsThe PiCV cap gene was cloned and fused with different fusion partners including a His-tag, a GST-tag (glutathioine-S-transferase tag) and a Trx-His-tag (thioredoxin-His tag). The resulting constructs were then expressed after transformation into a number of different E. coli strains; these then had their protein expression evaluated. The expression of the recombinant Cap protein in E. coli was significantly increased when Cap protein was fused with either a GST-tag or a Trx-His tag rather than a His-tag. After various rare amino acid codons presented in the Cap protein were optimized to give the sequence rCapopt, the expression level of the GST-rCapopt in E. coli BL21(DE3) was further increased to a significant degree. The highest protein expression level of GST-rCapopt obtained was 394.27 ± 26.1 mg/L per liter using the E. coli strain BL21(DE3)-pLysS. Moreover, approximately 74.5% of the expressed GST-rCapopt was in soluble form, which is higher than the soluble Trx-His-rCapopt expressed using the BL21(DE3)-pLysS strain. After purification using a GST affinity column combined with ion-exchange chromatography, the purified recombinant GST-rCapopt protein was found to have good antigenic activity when tested against PiCV-infected pigeon sera.ConclusionsThese findings shows that the E. coli-expressed full-length PiCV Cap protein has great potential in terms of large-scaled production and this should allow in the future the development of a serodiagnostic kit that is able to clinically detect PiCV infection in pigeons.

Immunogenicity of Pigeon Circovirus Recombinant Capsid Protein in Pigeons

“…Currently, gene-engineered vaccines based on the major immunogens like capsid proteins of viruses are under development in both human and veterinary medicine. In recent years, recombinant capsid proteins of various circoviruses like DuCV, PCV2, PBFDV and PiCV have been produced in bacterial, yeast, and baculovirus systems [ 10 , 26 , 29 , 30 , 31 , 32 , 33 , 34 , 39 , 40 , 41 ]. Little is known, however, about the immunogenicity of avian circoviruses’ recombinant capsid proteins.…”

“…Traditional vaccines are mainly composed of live, attenuated, or inactivated pathogens; however, no laboratory protocol has so far been developed for culturing PiCV, and for this reason, the specific immunoprophylaxis of PiCV infections in pigeons is impossible [ 28 ]. Various methods for antigen production for diagnostic tests—PiCV recombinant capsid protein (rCP)—have been developed in recent years [ 10 , 28 , 29 , 30 ]. PiCV rCP could also be a potential candidate as a vaccine antigen, but its immunogenicity has not been confirmed in scientific research.…”

The epidemiology, molecular characterization and clinical pathology of circovirus infections in pigeons – current knowledge

“…A specific protocol for the prevention of PiCV infections in pigeons has not yet been developed due to the absence of vaccines targeting this pathogen. In recent years, attempts have been made to develop a recombinant PiCV capsid protein for diagnostic tests and a subunit vaccine against the PiCV (Daum et al 2009;Lai et al 2014;Stenzel et al 2016). This vaccine could protect pigeons against PiCV infection and, possibly, lower the prevalence of YPDS.…”

Generation of a Library of Carbohydrate-Active Enzymes for Plant Biomass Deconstruction

“…The data, presented in Figure 3 B, revealed that lower numbers of targets were obtained from genes synthesized artificially ( p = 0.04). Several studies have demonstrated that codon optimization for heterologous production in E. coli can potentially increase recombinant protein expression, especially in sequences of eukaryotic and archaeal origin [ 23 , 24 , 25 , 26 ]. However, other studies revealed that E. coli codon optimization may lead to overexpression of recombinant proteins, thus resulting in a higher tendency to generate insoluble inclusion bodies [ 27 , 28 ].…”