Heterologous expression and secretion of a Streptomyces scabies esterase in Streptomyces lividans and Escherichia coli.
Heterologous expression and secretion of a Streptomyces scabies esterase in Streptomyces lividans and Escherichia coli.
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浅青紫链霉菌和大肠杆菌中链霉菌疥疮酯酶的异源表达和分泌。
DOI：
10.1128/jb.174.8.2431-2439.1992
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发表时间：
1992
影响因子：
3.2
通讯作者：
Schottel,JL
中科院分区：
文献类型：
--
作者：
Hale,V;McGrew,M;Carlson,B;Schottel,JL
从疥疮链霉菌FL 1中克隆了酯酶基因，并将其在变铅青链霉菌质粒pIJ 486和pIJ 702中表达。In S.在变铅青藻中，酯酶基因在生长后期表达，并受锌的调控，如在S.疥疮从S.橄榄色。N-末端氨基酸序列分析表明，S.在S.疥疮Western印迹（免疫印迹）显示存在的40 kDa的前体形式的酯酶在细胞质提取物。将23个氨基酸的缺失引入到酯酶的推定信号序列中。当这种缺失型酯酶在S.在变青聚糖中，产生了一种细胞质38-kDa前体蛋白，但没有检测到分泌的酯酶，这表明缺失的序列对于有效加工和分泌的重要性。将酯酶基因克隆到大肠杆菌pUC 119质粒中。通过使用lac启动子序列，酯酶基因被表达，并且大部分酯酶定位于周质空间。
The esterase gene from Streptomyces scabies FL1 was cloned and expressed in Streptomyces lividans on plasmids pIJ486 and pIJ702. In S. lividans, the esterase gene was expressed during later stages of growth and was regulated by zinc, as is seen with S. scabies. The 36-kDa secreted form of the esterase was purified from S. lividans. N-terminal amino acid sequencing indicated that the processing site utilized in S. lividans for the removal of the signal sequence was the same as that recognized for processing in S. scabies. Western blots (immunoblots) revealed the presence of a 40-kDa precursor form of the esterase in cytoplasmic extracts. A 23-amino-acid deletion was introduced into the putative signal sequence for the esterase. When this deleted form of the esterase was expressed in S. lividans, a cytoplasmic 38-kDa precursor protein was produced but no secreted esterase was detected, suggesting the importance of the deleted sequence for efficient processing and secretion. The esterase gene was also cloned into the pUC119 plasmid in Escherichia coli. By using the lac promoter sequence, the esterase gene was expressed, and the majority of the esterase was localized to the periplasmic space.