期刊
JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY
卷 37, 期 4, 页码 349-361出版社
OXFORD UNIV PRESS
DOI: 10.1007/s10295-009-0680-2
关键词
Escherichia coli; Flagellum; Hemolysin; Lipase B; Protein secretion; Pseudozyma antarctica; Recombinant protein production
资金
- Natural Sciences and Engineering Research Council (NSERC) of Canada
- Canada Research Chair (CRC)
Functional expression of recombinant Pseudozyma antarctica lipase B (PalB) in Escherichia coli has been technically problematic due to protein misfolding, ineffective disulfide bond formation, and protein instability associated with intracellular proteolysis. To overcome these problems, an alternative approach was explored in this study by extracellular secretion of PalB via two Sec-independent secretion systems, i.e., the alpha-hemolysin (type I) and the modified flagellar (type III) secretion systems, which can export proteins of interest from the cytoplasm directly to the exterior of the cell. Both shaker flask and bioreactor cultivations were performed to characterize the developed PalB expression/secretion systems. Bioactive PalB was expressed and secreted extracellularly either as a HlyA fusion (i.e., PalB-HlyA via type I system) or an intact protein (via type III system). However, the secretion intermediates in the intracellular fraction of culture samples were non-bioactive even though they were soluble, suggesting that the extracellular secretion did mediate the development of PalB activity. Also importantly, the secretion strategy appeared to have a minimum impact on cell physiology. PalB secretion via the type I system was fast with higher specific PalB activities but poor cell growth. On the other hand, the secretion via the type III system was slow with lower specific PalB activities but effective cell growth.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据