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

作者：Lai Guan Hua; Lin Yen Chang; Tsai Yi Lun; Lien Yi Yang; Lin Ming Kuem; Chen Hsi Jien; Chang Wen Te; Tzen Jason T C^*; Lee Meng Shiou

来源：BMC Veterinary Research, 2014, 10(1): 115.

DOI：10.1186/1746-6148-10-115

Background: Pigeon 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. %26lt;br%26gt;Results: The 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 rCap(opt), the expression level of the GST-rCap(opt) in E. coli BL21(DE3) was further increased to a significant degree. The highest protein expression level of GST-rCap(opt) 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-rCap(opt) was in soluble form, which is higher than the soluble Trx-His-rCap(opt) expressed using the BL21(DE3)-pLysS strain. After purification using a GST affinity column combined with ion-exchange chromatography, the purified recombinant GST-rCap(opt) protein was found to have good antigenic activity when tested against PiCV-infected pigeon sera. %26lt;br%26gt;Conclusions: These 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.

• 出版日期2014-5-22
• 单位中国医科大学