Ishida Yojiro, Inouye Masayori
Department of Biochemistry and Cell Biology, Center for Advanced Biotechnology and Medicine, Rutgers-Robert Wood Johnson Medical School, 679 Hoes Lane West, Piscataway, NJ, 08854, USA.
AMB Express. 2016 Mar;6(1):19. doi: 10.1186/s13568-016-0190-3. Epub 2016 Mar 2.
Bac7 (1-35) is an Arg- and Pro-rich peptide antibiotic, produced in bovine cells to protect them from microbial infection. It has been demonstrated to inhibit the protein synthesis in E. coli, leading to cell death. Because of its toxicity, no cost effective methods have been developed for Bac7 production in Escherichia coli for its potential clinical use. Here, we found a method to suppress Bac7 (1-35) toxicity in E. coli to establish its high expression system, in which Bac7 (1-35) was fused to the C-terminal end of protein S, a major spore-coat protein from Myxococcus xanthus, using a linker containing a Factor Xa cleavage site. The resulting His6-PrS2-Bac7 (1-35) (PrS2 is consisted of two N-terminal half domains of protein S connected in tandem) was well expressed using the Single-Protein Production (SPP) system at low temperature and subsequently purified in a single step by using a Ni column. The combination of protein S fusion and its expression in the SPP system at low temperature appeared to suppress Bac7 (1-35) toxicity. Both the purified His6-PrS2-Bac7 (1-35) and His6-PrS2-Bac7 (1-35) treated by Factor Xa were proven to be a potent inhibitor for cell-free protein synthesis.
Bac7 (1-35)是一种富含精氨酸和脯氨酸的肽类抗生素，由牛细胞产生以保护其免受微生物感染。已证明它能抑制大肠杆菌中的蛋白质合成，导致细胞死亡。由于其毒性，尚未开发出具有成本效益的方法在大肠杆菌中生产Bac7以供其潜在临床使用。在此，我们发现了一种抑制Bac7 (1-35)在大肠杆菌中毒性的方法，以建立其高效表达系统，其中Bac7 (1-35)通过含有凝血因子Xa切割位点的接头与来自黄色粘球菌的主要芽孢衣蛋白S的C末端融合。使用单蛋白生产（SPP）系统在低温下可良好表达产生的His6-PrS2-Bac7 (1-35)（PrS2由串联连接的蛋白S的两个N末端半结构域组成），随后通过镍柱一步纯化。蛋白S融合及其在低温下的SPP系统中表达的组合似乎抑制了Bac7 (1-35)的毒性。经凝血因子Xa处理的纯化His6-PrS2-Bac7 (1-35)和His6-PrS2-Bac7 (1-35)均被证明是无细胞蛋白质合成的有效抑制剂。
