Abstract
Replacement of a specific amino acid residue in a protein with nonnatural analogues is highly challenging because of their cellular toxicity. We demonstrate for the first time the replacement of all arginine (Arg) residues in a protein with canavanine (Can), a toxic Arg analogue. All Arg residues in the 5-base specific (UACAU) mRNA interferase from Bacillus subtilis (MazF-bs(arg)) were replaced with Can by using the single-protein production system in Escherichia coli. The resulting MazF-bs(can) gained a 6-base recognition sequence, UACAUA, for RNA cleavage instead of the 5-base sequence, UACAU, for MazF-bs(arg). Mass spectrometry analysis confirmed that all Arg residues were replaced with Can. The present system offers a novel approach to create new functional proteins by replacing a specific amino acid in a protein with its analogues.
Highlights
Canavanine (Can) is a highly toxic arginine (Arg) analogue found in some plant seeds
In the Single Protein Production (SPP) system, E. coli cells are converted into a bioreactor producing only a target protein, in which an ACA-specific mRNA interferase, MazF-ec, from E. coli is induced to eliminate all cellular mRNAs but the ACA-less mRNA for the target protein
In MazF(⌬H), His-28 and Gly-27 in MazF-ec were replaced with Arg and Lys, respectively, which has no effect on the MazF-ec mRNA interferase activity (19)
Summary
Canavanine (Can) is a highly toxic arginine (Arg) analogue found in some plant seeds. It is quite intriguing to replace all residues of a specific amino acid in a protein with its analogues, because it may create novel functional proteins with altered structures. An orthogonal aminoacyl tRNA synthetase/tRNA pair from other species was incorporated into bacteria (4, 5) or eukaryotes (6) One such highly toxic analogue is L-canavanine (Can), an Arg analogue (Fig. 1, A and B) that is found as an insecticide in certain leguminous plants such as jack bean (7). It has been shown that mRNA interference is mediated by proteins using sequence-specific endoribonucleases, called mRNA interferases (13) The first such enzyme reported was MazF-ec from Escherichia coli consisting of 111 residues, which cleaves RNA at ACA sequences (14). We attempted to replace all 7 Arg residues in MazF-bs, a 5-base specific RNA interferase from Bacillus subtilis (18) with Can using the Single Protein Production (SPP) system (19, 20)
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