对一种新型枯草杆菌蛋白酶抑制剂基因及其突变衍生物在淡紫链霉菌中表达和分泌小鼠肿瘤坏死因子α的评估。
Evaluation of a novel subtilisin inhibitor gene and mutant derivatives for the expression and secretion of mouse tumor necrosis factor alpha by Streptomyces lividans.
作者信息
Lammertyn E, Van Mellaert L, Schacht S, Dillen C, Sablon E, Van Broekhoven A, Anné J
机构信息
Laboratory of Bacteriology, Rega Institute, Katholieke Universiteit Leuven, Belgium.
出版信息
Appl Environ Microbiol. 1997 May;63(5):1808-13. doi: 10.1128/aem.63.5.1808-1813.1997.
Abstract
In order to evaluate the expression and secretion signals of the highly secreted subtilisin inhibitor of Streptomyces venezuelae CBS762.70 (VSI) for the production of heterologous proteins by Streptomyces lividans, mouse tumor necrosis factor alpha (mTNF) was chosen as a model protein. The mTNF cDNA was fused to the vsi signal sequence. The analysis of secretion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and biological activity measurements revealed an efficient translocation of mTNF. Up to 300 mg of secreted biologically active mTNF per liter could be obtained in shaken-flask cultures. By analyzing the effects of mutations in the N region of the VSI signal peptide on secretion, we found that decreasing the +3 charge of the wild-type protein to +2 resulted in a 3- to 10-fold increase in secretion.
摘要
为了评估委内瑞拉链霉菌CBS762.70的高分泌枯草杆菌蛋白酶抑制剂（VSI）的表达和分泌信号，以便用于淡紫链霉菌生产异源蛋白，选择小鼠肿瘤坏死因子α（mTNF）作为模型蛋白。将mTNF cDNA与vsi信号序列融合。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析分泌情况并进行生物活性测量，结果表明mTNF能够有效转运。在摇瓶培养中，每升可获得高达300毫克分泌型生物活性mTNF。通过分析VSI信号肽N区域突变对分泌的影响，我们发现将野生型蛋白的+3电荷降低到+2会导致分泌增加3至10倍。
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