title: High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression.
abstract: 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.
  
  
  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 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.
  
  
  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.
authors.forename: Guan-Hua
authors.initials: GH
authors.lastname: Lai
authors.forename: Yen-Chang
authors.initials: YC
authors.lastname: Lin
authors.forename: Yi-Lun
authors.initials: YL
authors.lastname: Tsai
authors.forename: Yi-Yang
authors.initials: YY
authors.lastname: Lien
authors.forename: Ming-Kuem
authors.initials: MK
authors.lastname: Lin
authors.forename: Hsi-Jien
authors.initials: HJ
authors.lastname: Chen
authors.forename: Wen-Te
authors.initials: WT
authors.lastname: Chang
authors.affiliation: Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan. TCTZEN@dragon.nchu.edu.tw.
authors.forename: Jason T C
authors.initials: JT
authors.lastname: Tzen
authors.forename: Meng-Shiou
authors.initials: MS
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D002845:Chromatography; D017925:Circovirus / Q000145:classification* / Q000502:physiology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000502:physiology*; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D014779:Virus Replication / Q000502:physiology*
chemical_list: D036022:Capsid Proteins; D011994:Recombinant Proteins