Molecular cloning and expression of a synthetic DNA coding for the antimicrobial protein of bull seminal plasma

A DNA carrying the coding sequence for the antimicrobial protein from bull seminal plasma (SAP) was obtained by enzymic ligation of six synthetic oligonucleotides. The 162 bp synthetic DNA fragment was cloned into the C-terminal part of the lacZ-gene employing the vector pUR289. Expression in E. coli in the presence of the inducer isopropylthiogalactoside (IPTG) led to the formation of a fusion protein, which was shown by immuno-blotting to contain immuno-reactive antimicrobial protein. Approximately 90 min after induction, the cells stopped growing and the culture was found to contain no viable cells 3 h after induction. We conclude from this observation that the beta-galactosidase-antimicrobial protein fusion product was toxic for the E. coli cell and that the SAP-residue attached to beta-galactosidase was responsible for the cytotoxicity.