Isolation of soluble proteins from an industrial strain Streptomyces avermitilis in complex culture medium for two-dimensional gel electrophoresis.
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.
Yin
Peng
P
State Key Laboratory of Bioreactor Engineering, National Research Center for Biotechnology (Shanghai), East China University of Science and Technology, Shanghai 200237, PR China.
Wang
Yong-Hong
YH
Zhang
Si-Liang
SL
Chu
Ju
J
Zhuang
Ying-Ping
YP
Wang
Mei-Long
ML
Zhou
Jin
J
D000096:Acetone; D001426:Bacterial Proteins / Q000032:analysis* / Q000302:isolation & purification*; D015180:Electrophoresis, Gel, Two-Dimensional; D005590:Fractional Precipitation; D011489:Protein Denaturation; D020543:Proteome / Q000032:analysis* / Q000302:isolation & purification*; D013302:Streptomyces / Q000737:chemistry*; D014238:Trichloroacetic Acid
D001426:Bacterial Proteins; D020543:Proteome; D000096:Acetone; D014238:Trichloroacetic Acid