Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis
Novel plasmid-based expression vectors for intra- and extracellular production of recombinant proteins in Bacillus subtilis
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DOI：
10.1016/j.pep.2005.07.005
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发表时间：
2006-04-01
影响因子：
1.6
通讯作者：
Schumann, W
中科院分区：
文献类型：
--
作者：
Phan, TTP;Nguyen, HD;Schumann, W
已经构建了两种基于质粒的表达载体，其中一种允许重组蛋白质的细胞内生产，而第二种将蛋白质引导到培养基中。两种载体都使用枯草芽孢杆菌的groESL操纵子（编码必需的热休克蛋白GroES和GroEL）之前的强启动子，其融合到lac操纵子，允许通过添加ITPG进行诱导。虽然在不存在诱导剂的情况下这些表达盒的背景表达水平非常低，但测量到约1300的诱导因子。当基因hipG和pbpE（分别编码热休克蛋白和青霉素结合蛋白）与groE启动子融合时，加入IPTG后产生的重组蛋白的量分别占总细胞蛋白的10%和13%。为了获得重组蛋白的分泌，将编码来自解淀粉芽孢杆菌的α-淀粉酶的amyQ基因的信号肽的编码区与groE启动子融合。热纤梭菌分泌amyQ α-淀粉酶和纤维素酶A、B。(c)2005年爱思唯尔公司All rights reserved.
Two plasmid-based expression vectors have been constructed where one allows intracellular production of recombinant proteins while the second directs the proteins into the Culture medium. Both vectors use the strong promoter preceding the groESL operon (codes for the essential heat shock proteins GroES and GroEL) of Bacillus subtilis fused to the lac operator allowing their induction by addition of ITPG. While the background level of expression of these expression cassettes is very low in the absence of the inducer, an induction factor of about 1300 was measured. When the genes hipG and pbpE (coding for a heat shock protein and a penicillin-binding protein, respectively) were fused to the groE promoter, the amount of recombinant protein produced after addition of IPTG represented 10 and 13%, respectively, of the total cellular protein. To obtain secretion of recombinant proteins, the coding region for the signal peptide of the amyQ gene encoding an alpha-amylase from Bacillus amyloliquefasciens was fused to the groE promoter. High-level secretion of amyQ alpha-arnylase and cellulase A and B of Clostridium thermocellum was demonstrated. (c) 2005 Elsevier Inc. All rights reserved.