Use of amino acids as inducers for high-level protein expression in the single-protein production system.
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 to the unlabeled target protein.
Vaiphei
S Thangminlal
ST
Department of Biochemistry, Robert Wood Johnson Medical School, and Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854, USA.
Mao
Lili
L
Shimazu
Tsutomu
T
Park
Jung-Ho
JH
Inouye
Masayori
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D019149:Bioreactors / Q000382:microbiology*; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D004722:Endoribonucleases / Q000096:biosynthesis*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis*; D007544:Isopropyl Thiogalactoside; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis / Q000502:physiology*; D015202:Protein Engineering / Q000379:methods*; D017422:Sequence Analysis, DNA; D014364:Tryptophan
D017931:DNA Primers; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C422955:MazF protein, E coli; D007544:Isopropyl Thiogalactoside; D014364:Tryptophan; D004722:Endoribonucleases