Use of Amino Acids as Inducers for High-Level Protein Expression in the Single-Protein Production System

Abstract

By taking advantage of MazF, an ACA codon-specific mRNA interferase, Escherichia coli cells can be converted into a bioreactor producing only a single protein of interest by using an ACA-less mRNA for the protein. In this single-protein production (SPP) system, we engineered MazF by replacing two tryptophan residues in positions 14 and 83 with Phe (W14F) and Leu (W83L), respectively. Upon the addition of an inducer (IPTG [isopropyl-␤-D-thiogalactopyranoside]), the mutated MazF [MazF(⌬W)] can still be produced even in the absence of tryptophan in the medium by using a Trp auxotroph, while a target protein having Trp residues cannot be produced. However, at 3 h after the addition of IPTG, the addition of tryptophan to the medium exclusively induces production of the target protein at a high level. A similar SPP system was also constructed with the use of a His-less protein [MazF(⌬H)] and a His auxotroph. Using these dual-induction systems, isotopic enrichments of 13 C, 15 N, and 2 H were highly improved by almost complete suppression of the production of the unlabeled target protein. In both systems, isotopic incorporation reached more than 98% labeling efficiency, significantly reducing the background attributable to the unlabeled target protein.

A safety type genetically engineered bacterium with red fluorescence which can be used to degrade organophosphorus pesticides

“…Usually, the expression of target proteins in GEMs requires an inducer (Menzella and Gramajo 2004;Vaiphei et al 2010;Kim et al 2012), which places an additional cost on the expression of the target protein, thereby reducing the GEMs cost-effectiveness. However, by cloning the target gene downstream of the lambda P L promoter, deleting the copy of the lambda cIts857 repressor from the plasmid pBV220 and choosing an appropriate host, we constructed a constitutive protein expression unit.…”

Replacement of All Arginine Residues with Canavanine in MazF-bs mRNA Interferase Changes Its Specificity

“…Strain Construction-E. coli BL21(DE3) (⌬argH⌬trpC⌬hisB) was constructed from E. coli BL21(DE3) (⌬trpC⌬hisB) (19) by P1 transduction using the ⌬argH strain from the Keio collection (21).…”

“…The gene was designed for the optimal codon usage in E. coli and to have no ACA sequences. The gene was cloned into pColdIII (SP-4) (19,20).…”

“…by the SPP System-To replace all 7 Arg residues in MazF-bs with Can, we applied the SPP system (19,20) with use of an Arg auxotroph. In the 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.…”

“…For the complete replacement of all Arg residues with Can, it is also important to block the biosynthesis of Arg in the cells using an Arg auxotroph. For this, the argH deletion mutation from the Keio collection (21) was transduced into E. coli BL21(DE3) ⌬trpC, ⌬hisB (19). The gene for MazF-bs designed to be ACA-less and codon-optimized for E. coli ( Fig.…”