Expression, purification, and bioactivity of GST-fused v-Src from a bacterial expression system

• Published in: Journal of Zhejiang University. B. Science Vol. 7; no. 1; pp. 13 - 19
• Main Author: 龚兴国 纪静 谢捷 周远 张俊彦 钟文涛
• Format: Journal Article
• Language: English
• Published: China Springer Nature B.V 2006; Institute of Biomacromolecule and Enzyme Engineering, School of Life Sciences, Zhejiang University, Hangzhou 310027, China; Zhejiang University Press
• Subjects: Bacteria; Bacterial Proteins - biosynthesis; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Biotechnology & Food Sciences; Glutathione Transferase - biosynthesis; Glutathione Transferase - genetics; Glutathione Transferase - isolation & purification; Kinases; Oncogene Protein pp60(v-src) - biosynthesis; Oncogene Protein pp60(v-src) - chemistry; Oncogene Protein pp60(v-src) - genetics; Oncogene Protein pp60(v-src) - isolation & purification; Protein Engineering - methods; Proteins; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - isolation & purification; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Signal transduction; Protein tyrosine kinase; v-Src; Inclusion body; Orthogonalization; GST-fusion
• ISSN: 1673-1581; 1862-1783
• DOI: 10.1631/jzus.2006.B0013

v-Src is a non-receptor protein tyrosine kinase involved in many signal transduction pathways and closely related to the activation and development of cancers. We present here the expression, purification, and bioactivity of a GST （glutathione S-transferase）-fused v-Src from a bacterial expression system. Different culture conditions were examined in an isopropyl ,B-D-thiogalactopyranoside （IPTG）-regulated expression, and the fused protein was purified using GSH （glutathione） affinity chromatography. ELISA （enzyme-linked immunosorbent assay） was employed to determine the phosphorylation kinase activity of the GST-fused v-Src. This strategy seems to be more promising than the insect cell system or other eukaryotic systems employed in earlier Src expression.