Use of amino acids as inducers for high-level protein expression in the single-protein production system.

Applied and Environmental Microbiology
S Thangminlal VaipheiMasayori Inouye

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-beta-d-thiogalactopyranoside]), the mutated MazF [MazF(DeltaW)] 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(DeltaH)] 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 ...Continue Reading

References

Jun 1, 1993·Proceedings of the National Academy of Sciences of the United States of America·C P HillD Eisenberg
Oct 9, 2002·Genes & Development·Andrea Cuconati, Eileen White
Apr 20, 2005·Molecular Cell·Motoo SuzukiMasayori Inouye
Oct 6, 2006·The Journal of Biological Chemistry·Motoo SuzukiMasayori Inouye
Jul 21, 2007·Nature Protocols·Motoo SuzukiMasayori Inouye
Oct 27, 2009·Journal of Structural and Functional Genomics·Lili MaoMasayori Inouye
Mar 25, 2010·Journal of Structural and Functional Genomics·William M SchneiderGaetano T Montelione

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Citations

Feb 5, 2013·The Journal of Biological Chemistry·Yojiro IshidaMasayori Inouye
Aug 9, 2011·Critical Reviews in Biochemistry and Molecular Biology·Finbarr Hayes, Laurence Van Melderen
Sep 11, 2018·Biotechnology and Bioengineering·Sen YangZhen Kang
Feb 9, 2019·Journal, Genetic Engineering & Biotechnology·Padikara K SatheeshkumarPerumana R Sudhakaran

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