An efficient co-expression and purification system for the complex of Stx4 and C-terminal domain of Synip

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• 作者：Wei Tian ; Cong Ma ; Yingfang Liu ; Tao Xu
• 关键词：GLUT4 vesicle ; SynipCT– ; Stx4 complex ; In vitro pull-down ; Co-expression ; Purification ; Gel-filtration analysis ; Mass identification
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：2008
• 出版时间：4 July 2008
• 年：2008
• 卷：371
• 期：3
• 页码：366-370
• 全文大小：1009 K

文摘

Synip and Stx4 complex plays a key role in GLUT4 vesicle trafficking and fusion with plasma membrane. The interaction of Synip with Stx4 prevents interaction of VAMP2 located in GLUT4 vesicle with Stx4 in basal state. Insulin induces the dissociation of the Synip and Stx4 complex, and then triggers VAMP2 to interact with Stx4 to form the SNARE complex, thus promoting the vesicle fusion. In this report, we adopt a novel system for co-expression of the Synip and Stx4 by using two common vectors pGEX6p-1 and pET28a(+) to investigate their expression, purification, and interaction. Through this co-expression system, we successfully co-expressed the Synip and Stx4 complex with high yield, and co-purified at an approximate 1:1 molar ratio with high purity (95 % ). We also demonstrate that the 1–28 residues of Stx4 are dispensable for interaction with Synip using this co-expression system.