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

文化大學機構典藏 CCUR > 環境規劃暨生物資源學院 > 農學院 > 生物科技研究所 > 期刊論文 > Item 987654321/29203

jsp.display-item.identifier=請使用永久網址來引用或連結此文件: https://irlib.pccu.edu.tw/handle/987654321/29203

题名:	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
贡献者:	生物科技研究所
关键词:	POLYMERASE-CHAIN-REACTION CHICKEN ANEMIA VIRUS ESCHERICHIA-COLI FEATHER DISEASE INFECTION DIAGNOSIS ANTIBODY VACCINE CLONING TESTS
日期:	2014-05-22
上传时间:	2015-01-26 11:06:05 (UTC+8)
摘要:	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 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. 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.
關聯:	BMC VETERINARY RESEARCH 卷: 10 文獻號碼: 115
显示于类别:	[生物科技研究所 ] 期刊論文

文件中的档案:

档案	描述	大小	格式	浏览次数
index.html		0Kb	HTML	324	检视/开启

检视Licence

在CCUR中所有的数据项都受到原著作权保护.

数据加载中.....