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 to the unlabeled target protein.
journal_name
Appl Environ Microbioljournal_title
Applied and environmental microbiologyauthors
Vaiphei ST,Mao L,Shimazu T,Park JH,Inouye Mdoi
10.1128/AEM.00815-10subject
Has Abstractpub_date
2010-09-01 00:00:00pages
6063-8issue
18eissn
0099-2240issn
1098-5336pii
AEM.00815-10journal_volume
76pub_type
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