| abstract |
The method by which polypeptides having residues other than the 20 common amino acids are made is established. A leader peptide sequence (SEQ ID NO: 7) and its gene are identified which induce or assist post-translational modifications of Cys, Thr and Ser in prokaryotes. The leader sequence may be used to induce the presence of covalent bonding sites in polypeptides and can be expressed by either naturally occurring or artificial means. Further, a subtilin mutant substituting isoleucine for Glu4 of the native sequence exhibits a 57-fold improvement in stability, resisting modification of the dehydroalanine residue at position 5. This stable mutant exhibits 3-4 times the specific activity, in suppression of bacterial spore outgrowth, of the native bacteriocin. A method for site-specific mutagenesis, as well as the resulting mutant gene, plasmid and transformant is similarly set forth. |