Expression, purification, and characterization of soluble RpfD with high bioactivity as a recombinant protein in Mycobacterium vaccae
Expression, purification, and characterization of soluble RpfD with high bioactivity as a recombinant protein in Mycobacterium vaccae
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具有高生物活性的可溶性 RpfD 在母牛分枝杆菌中的表达、纯化和表征
DOI：
10.1016/j.pep.2007.05.002
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发表时间：
2007-09-01
影响因子：
1.6
通讯作者：
Xu, Zhikai
中科院分区：
文献类型：
--
作者：
Gao, Hui;Bai, Yinlan;Xu, Zhikai
复苏促进因子D（Resuscitation promoting factor D，RpfD）是结核分枝杆菌Rpf-like家族的5个成员之一，均具有促进复苏的活性。在此，扩增了完整的rpfD基因，并将其克隆到pDE 22表达载体中。然后在母牛分枝杆菌中表达可溶性RpfD的重组形式（sRpfD）。可溶性重组RpfD通过Ni-Sepharose亲和层析纯化。纯化的最终产物纯度>95%，并且通过考马斯亮蓝R-250染色的15% SDS-PAGE测定分子量为24.0kDa。纯化收率约为1。10 mg/L的培养物。通过刺激无毒M.结核病H37 Ra，处于“不可培养”状态。结果表明，M.母牛菌苗能更有效地刺激M.结核杆菌H37 Ra的促复苏作用强于大肠杆菌DH 5 α的复性重组RpfD（iRpfD），兔抗sRpfD血清能完全抑制这两种重组RpfD的促复苏作用。我们的研究表明，该表达系统可以促进大规模生产和纯化的sRpfD具有较高的生物活性，为进一步的功能，药理学和临床研究。(C)2007年爱思唯尔公司All rights reserved.
Resuscitation promoting factor D (RpfD) is one of the five members of the Rpf-like family in Mycobacterium tuberculosis, which all have the resuscitation-promoting activity. Here, the complete rpfD gene was amplified and cloned into pDE22 expression vector. Then a recombinant form of soluble RpfD (sRpfD) was expressed in Mycobacterium vaccae. The soluble recombinant RpfD was purified by Ni-Sepharose affinity chromatography. The purified final product was >95% pure, and the molecular weight was 24.0 kDa, determined by 15% SDS-PAGE stained with Coomassie brilliant blue R-250. The yield of purification was about 1. 10 mg/L of the culture. The biochemical property of the sRpfD was analyzed by stimulating the resuscitation of avirulent M. tuberculosis H37Ra which was in "non-culturable" condition. The results indicate that the sRpfD from M. vaccae could more efficiently stimulate the resuscitation of M. tuberculosis H37Ra than the refolded recombinant RpfD (iRpfD) from Escherichia coli DH5 alpha, and that the rabbit anti- sRpfD serum could completely inhibit this resuscitation-promoting effect caused by these two kinds of recombinant RpfD. Our study indicates that this expression system may facilitate large-scale production and purification of sRpfD which have high biological activity for further functional, pharmacological and clinical investigations. (C) 2007 Elsevier Inc. All rights reserved.