title: Suppression of the toxicity of Bac7 (1-35), a bovine peptide antibiotic, and its production in E. coli.
abstract: 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.
authors.affiliation: 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.
authors.forename: Yojiro
authors.initials: Y
authors.lastname: Ishida
authors.affiliation: 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. inouye@cabm.rutgers.edu.
authors.forename: Masayori
authors.initials: M
authors.lastname: Inouye
keywords: Antimicrobial peptide; Bac7 (1–35); Bovine; E. coli; Protein S; SPP system