在大肠杆菌中表达的重组人粒细胞-巨噬细胞集落刺激因子的纯化

Purification of recombinant human granulocyte-macrophage colony stimulating factor expressed in Escherichia coli.

作者信息

Ling M, Zou M, Xu M, Wang J, Ma X

机构信息

Beijing Institute of Basic Medical Sciences, China.

出版信息

Chin J Biotechnol. 1995;11(3):157-62.

PMID:8679931

Abstract

Recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) was expressed as inclusion bodies (IB) in E. coli. A simple and effective protocol has been worked out for the purification. IB collected after the breakage of bacteria through sonication were subjected to repeated washing followed by solubilization in TE buffer (50 mmol/L of Tris.HCl, 1 mmol/L of EDTA, pH 8.3) containing 8 mol/L urea and 10 mmol/L DL-dithiothreitol. By means of Sephacryl-200 HR, refolding, and Q Sepharose Fast Flow, rhGM-CSF was obtained with a purity of 99%. The total protein recovery was 10% and specific activity of rhGM-CSF was 1 x 10(7) u/mg. The sequence of N-terminal 16 amino acid residues of purified rhGM-CSF was determined and found to be identical to the native protein. This study provided useful parameters for mass production of rhGM-CSF.

摘要

重组人粒细胞巨噬细胞集落刺激因子（rhGM-CSF）在大肠杆菌中以包涵体（IB）形式表达。已制定出一种简单有效的纯化方案。通过超声破碎细菌后收集的包涵体，先进行反复洗涤，然后溶解于含有8 mol/L尿素和10 mmol/L DL-二硫苏糖醇的TE缓冲液（50 mmol/L Tris.HCl，1 mmol/L EDTA，pH 8.3）中。通过Sephacryl-200 HR、复性和Q Sepharose Fast Flow，获得了纯度为99%的rhGM-CSF。总蛋白回收率为10%，rhGM-CSF的比活性为1×10⁷ u/mg。测定了纯化的rhGM-CSF N端16个氨基酸残基的序列，发现与天然蛋白相同。本研究为rhGM-CSF的大规模生产提供了有用的参数。