Tian Zi-gang, Dong Tian-tang, Yang Ya-lin, Teng Da, Wang Jian-hua
Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun Nandajie St., Haidian District, Beijing 100081, People's Republic of China.
Appl Microbiol Biotechnol. 2009 May;83(1):143-9. doi: 10.1007/s00253-009-1893-z. Epub 2009 Feb 10.
The tandem repeats of LFB15(W4,10)-HP(4-16) (LH) gene were cloned into vector pET32a(+) for recombinant expression in Escherichia coli. The E. coli C43(DE3) was successfully used as the expression host to avoid the cell death during induction in E. coli BL21(DE3). Fusion LH dimer was expressed as inclusion body at a portion of 35% of total cell protein and could be well purified by Ni(2+)-chelating chromatography. The recombinant LH was released by the cleavage of 50% formic acid, and its yield reached 11.3 mg/l with purity of 95%. The MIC(50) of 3.6 and 1.9 microM of recombinant LH against E. coli CMCC 44102 and Bacillus subtilis ATCC 6633 were determined, respectively. The results demonstrated that expression of tandem LH gene in E. coli C43(DE3) and formic acid cleavage would provide a potent efficient platform for the production of interested peptides.
将LFB15(W4,10)-HP(4 - 16)（LH）基因的串联重复序列克隆到载体pET32a(+)中，以便在大肠杆菌中进行重组表达。大肠杆菌C43(DE3)成功用作表达宿主，避免了在大肠杆菌BL21(DE3)诱导过程中的细胞死亡。融合的LH二聚体以包涵体形式表达，占总细胞蛋白的35%，并且可以通过镍离子螯合层析法很好地纯化。重组LH通过50%甲酸裂解释放，其产量达到11.3 mg/l，纯度为95%。分别测定了重组LH对大肠杆菌CMCC 44102和枯草芽孢杆菌ATCC 6633的最低抑菌浓度（MIC(50)），分别为3.6 μM和1.9 μM。结果表明，在大肠杆菌C43(DE3)中表达串联LH基因并通过甲酸裂解将为生产感兴趣的肽提供一个高效的平台。
