文献类型：期刊文献

英文题名：Isolation of soluble proteins from an industrial strain Streptomyces avermitilis in complex culture medium for two-dimensional gel electrophoresis

作者：Yin, Peng[1];Wang, Yong-Hong[1];Zhang, Si-Liang[1];Chu, Ju[1];Zhuang, Ying-Ping[1];Wang, Mei-Long[1];Zhou, Jin[1]

机构：[1]E China Univ Sci & Technol, Natl Res Ctr Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China

年份：2008

卷号：73

期号：2

起止页码：105

外文期刊名：JOURNAL OF MICROBIOLOGICAL METHODS

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000256165200005)】；

语种：英文

外文关键词：Streptomyces avermitilis; proteome; urea; thiourea; two-dimensional gel electrophoresis

摘要：Two-dimensional gel electrophoresis (2-DE) is a core proteomic technique to study protein expression and function in living organisms. Although 2-DE has been extensively used for the investigation of bacteria, yeast, animal and plant tissue cells, the isolation of proteins from the organisms and elimination of salt, nucleotide, polysaccharide, lipids and other contaminations from the samples often limit its application. In this study, the protocol for protein isolation from cells of Streptomyces avermitilis cultivated in partially insoluble complex medium was investigated. The goal was to make the obtained extraction samples suitable for the two-dimensional electrophoresis, thus make the further proteome analysis possible. Compared to non-denatured procedure, the denatured one, precipitating with 10% TCA in acetone, efficiently eliminated the interference substances from the cell lysate. Thiourea in the rehydration solution enhanced the resolubilization of protein pellets but led to heavy horizontal streaking in the 2-DE gels. High protein loading amount improved the resolution of some low abundance proteins but did not adapt to the high abundance proteins. And it was also important to collect cells at appropriate culture time according to the analysis target. With the optimized protein extraction protocol, the protein expression patterns of S. avermitilis during the onset of avermectin production in complex medium were analyzed. (c) 2008 Elsevier B.V. All rights reserved.