Expression and purification of full-length anti-apoptotic Bcl-2 using cell-free protein synthesis

被引：12

作者：

Pedersen, Anders ^{[2
]}

Wallgren, Marcus ^{[1
]}

Karlsson, B. Goran ^{[2
]}

Grobner, Gerhard ^{[1
]}

机构：

[1] Umea Univ, Dept Chem, SE-90187 Umea, Sweden

[2] Univ Gothenburg, Swedish NMR Ctr, SE-40530 Gothenburg, Sweden

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2011年 / 77卷 / 02期

基金：

瑞典研究理事会;

关键词：

Apoptosis; Bcl-2; Cell-free protein synthesis; Full-length protein; Optimized gene; MEMBRANE-PROTEINS; FAMILY; CHANNEL; CANCER; SWITCH; DEATH;

D O I：

10.1016/j.pep.2011.02.003

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The anti-apoptotic B cell CLL/lymphoma-2 (Bcl-2) protein is a key player in the regulation of programmed cell death and is linked to various types of cancer and their resistance to drug treatment. Biophysical and structural studies of the full-length intact Bcl-2 have been hampered due to difficulties in expression and severe solubility problems, precluding isolation of this hydrophobic membrane protein. Therefore, previous work has so far mainly been carried out using structurally modified Bcl-2 variants, lacking the transmembrane region. Thus, biophysical information regarding the full-length protein is still missing. Here, a protocol is presented for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)). A batch-based cell-free expression system, using extract isolated from Escherichia coli (E. coli) was employed to produce recombinant protein encoded by an optimized gene sequence. Presence of polyoxyethylene-(20)-cetyl-ether (Brij-58) in the reaction mixture and subsequently in the immobilized metal-affinity purification steps was crucial to keep Bcl-2(2) soluble. The obtained yield was 0.25-0.3 mg per ml of cell-free reaction. Far-UV circular dichroism (CD) spectroscopy confirmed the alpha-helical structure of the purified protein, characteristic for members of the Bcl-2 protein family. (c) 2011 Elsevier Inc. All rights reserved.

引用

页码：220 / 223

页数：4

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