期刊
MOLECULAR BIOTECHNOLOGY
卷 38, 期 3, 页码 221-232出版社
HUMANA PRESS INC
DOI: 10.1007/s12033-007-9018-3
关键词
AT-rich codon; codon optimization; high-level expression; recombinant human G-CSF; site-directed mutagenesis; specific product yield
Enhanced therapeutic importance of recombinant human granulocyte colony stimulating factor (rhG-CSF) has encouraged us to develop a processing method for its high-level expression in E. coli. In this study, we established a high-yielding clone by incorporation of silent mutations at N-terminal region of human G-CSF gene. We studied and optimized various parameters of culture conditions connected with the expression of rhG-CSF. The maximum expression was obtained in a defined medium supplemented with 1% glucose. The gene in pET-3a vector in E. coli BL21 (DE3) PLysS host strain was induced with 2 mM isopropyl beta-D-1-thiogalacto pyronoside. The cell growth and productivity was enhanced about 1.6- and 1.5-folds, respectively when inducing the culture at OD600 value of 6 than 2. The protein expression was significantly increased by addition of rifampicin at concentration of 200 mu g/ml. The AT content of 51.8% with suitable codon sequences at N-terminal region and the concentration of rifampicin were identified as the key factors with a significant impact on protein expression. The specific productivity of 104 mg/OD/l (68.7% of total cellular protein) of rhG-CSF was obtained toward the end of the study, which is almost 1.5 times higher yield than reported so far in the literature.
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