Molecular cloning, overexpression, and an efficient one-step purification of α5β1 integrin

被引：2

作者：

Tartaglia, Lawrence J. ^{[1
]}

Bennett, Antonette ^{[1
]}

Plattner, Alexander S. ^{[1
]}

Muzyczka, Nicholas ^{[2
]}

Ling, Chen ^{[3
]}

Srivastava, Arun ^{[3
]}

Agbandje-McKenna, Mavis ^{[1
]}

机构：

[1] Univ Florida, Coll Med, Dept Biochem & Mol Biol, Gainesville, FL 32610 USA

[2] Univ Florida, Coll Med, Dept Mol Genet & Microbiol, Gainesville, FL 32610 USA

[3] Univ Florida, Coll Med, Dept Genet, Gainesville, FL 32610 USA

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2013年 / 92卷 / 01期

关键词：

alpha 5 beta 1 Integrin expression; pFastBac1; vector; Recombinant baculovirus; Electron microscopy; ADENOASSOCIATED VIRUS; STATISTICAL-MODEL; CELL-ADHESION; EXPRESSION; ACTIVATION; PROTEINS;

D O I：

10.1016/j.pep.2013.08.013

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The alpha 5 beta 1 integrin heterodimer is involved in many cellular processes and is an anti-cancer therapeutic target. Therefore, access to quantities of protein suitable for studies aimed at understanding its biological functions is important. To this end, a large-scale protein expression system, utilizing the recombinant baculovirus/SF9 insect cell expression system, was created to produce the extracellular domain of the alpha 5 beta 1 integrin. An incorporated 8X-histidine tag enabled one-step nickel-column purification. Following sequence confirmation by LC-MS/MS, the conformation of the heterodimer was characterized by native dot blot and negative stain electron microscopy. Cellular transduction inhibition studies confirmed biological activity. The system allows expression and purification of alpha 5 beta 1 integrin in quantities suitable for an array of different experiments including structural biology. (C) 2013 Elsevier Inc. All rights reserved.

引用

页码：21 / 28

页数：8

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