Creation of a New protein by substituting all arginine residues by its toxic analogue, canavanine

Abstract

Complete substitution of a specific amino acid in a protein with its analogue may create a novel protein with unprecedented properties but is quite challenging, particularly if the analogue is highly toxic to living cells. Canavanine (Can), an analogue of Arginine (Arg) found in certain leguminous plants is highly toxic, in which a ε‐methylene group in Arg is replaced with an oxa group, lowering the pKa value from 12.48 to 7.04. Here, we demonstrate for the first time the complete replacement of Arg residues in a protein with Can, as all the seven Arg residues in an mRNA interferase from Bacillus subtilis, MazF‐bs were replaced with Can. To achieve this, the ACA‐less gene was engineered to produce only MazF‐bs in the presence of Can in a condensed culture of the Single‐Protein Production (SPP) system using an E. coli Arg‐auxotroph strain. We show that all Arg residues were completely replaced with Can as judged by mass spectroscopy. The CD spectrum of MazF‐bs(Can) was almost identical to that of the wild‐type MazF‐bs(Arg) but MazF‐bs(Can) was less thermo‐stable than MazF‐bs(Arg). Interestingly, MazF‐bs(Can) cleaved RNA not only at the original UUACA cleavage sites, but also at the CCACU sites. This is the first report to show that the complete replacement of Arginine with Canavanine in a protein, resulting in acquiring of a new function.