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Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis.

ABSTRACT: To develop a simple, speedy, economical and widely applicable method for multiple-site mutagenesis, we have substantially modified the Quik-Change Site-Directed Mutagenesis Kit protocol (Stratagene, La Jolla, CA). Our new protocol consists of (i) a PCR reaction using an in vitro technique, LDA (ligation-during-amplification), (ii) a DPN:I treatment to digest parental DNA and to make megaprimers and (iii) a synthesis of double-stranded plasmid DNA for bacterial transformation. While the Quik Change Kit protocol introduces mutations at a single site, requiring two complementary mutagenic oligonucleotides, our new protocol requires only one mutagenic oligonucleotide for a mutation site, and can introduce mutations in a plasmid at multiple sites simultaneously. A targeting efficiency >70% was consistently achieved for multiple-site mutagenesis. Furthermore, the new protocol allows random mutagenesis with degenerative primers, because it does not use two complementary primers. Our mutagenesis strategy was successfully used to alter the fluorescence properties of green fluorescent protein (GFP), creating a new-color GFP mutant, cyan-green fluorescent protein (CGFP). An eminent feature of CGFP is its remarkable stability in a wide pH range (pH 4-12). The use of CGFP would allow us to monitor protein localization quantitatively in acidic organelles in secretory pathways.

SUBMITTER: Sawano A

PROVIDER: S-EPMC108465 | biostudies-literature | 2000 Aug

REPOSITORIES: biostudies-literature

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Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis.

Sawano A A Miyawaki A A

Nucleic acids research 20000801 16

To develop a simple, speedy, economical and widely applicable method for multiple-site mutagenesis, we have substantially modified the Quik-Change Site-Directed Mutagenesis Kit protocol (Stratagene, La Jolla, CA). Our new protocol consists of (i) a PCR reaction using an in vitro technique, LDA (ligation-during-amplification), (ii) a DPN:I treatment to digest parental DNA and to make megaprimers and (iii) a synthesis of double-stranded plasmid DNA for bacterial transformation. While the Quik Chan ...[more]

PMID: 10931937

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Project description:BACKGROUND:Lycopene is a terpenoid pigment that has diverse applications in the food and medicine industries. A prospective approach for lycopene production is by metabolic engineering in microbial hosts, such as Escherichia coli. Isopentenyl diphosphate isomerase (IDI, E.C. 5.3.3.2) is one of the rate-limiting enzymes in the lycopene biosynthetic pathway and one major target during metabolic engineering. The properties of IDIs differ depending on the sources, but under physiological conditions, IDIs are limited by low enzyme activity, short half-life and weak substrate affinity. Therefore, it is important to prepare an excellent IDI by protein engineering. RESULTS:Directed evolution strategy (error-prone PCR) was utilized to optimize the activity of Saccharomyces cerevisiae IDI. Using three rounds of error-prone PCR; screening the development of a lycopene-dependent color reaction; and combinatorial site-specific saturation mutagenesis, three activity-enhancing mutations were identified: L141H, Y195F, and W256C. L141H, located near the active pocket inside the tertiary structure of IDI, formed a hydrogen bond with nearby ?-phosphates of isopentenylpyrophosphate (IPP). Phe-195 and Cys-256 were nonpolar amino acids and located near the hydrophobic group of IPP, enlarging the hydrophobic scope, and the active pocket indirectly. Purified IDI was characterized and the result showed that the Km of mutant IDI decreased by 10% compared with Km of the parent IDI, and Kcat was 28% fold improved compared to that of the original IDI. Results of a fermentation experiment revealed that mutant IDI had a 1.8-fold increased lycopene production and a 2.1-fold increased yield capacity compared to wild-type IDI. CONCLUSION:We prepared an engineered variant of IDI with improved catalytic activity by combining random and site directed mutagenesis. The best mutants produced by this approach enhanced catalytic activity while also displaying improved stability in pH, enhanced thermostability and longer half-life. Importantly, the mutant IDI could play an important role in fed-batch fermentation, being an effective and attractive biocatalyst for the production of biochemicals.

Dataset Information

Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis.

Publications

Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis.

Similar Datasets

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