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Semi-rational engineering of a coral fluorescent protein into an efficient highlighter.

ABSTRACT: Kaede is a natural photoconvertible fluorescent protein found in the coral Trachyphyllia geoffroyi. It contains a tripeptide, His 62-Tyr 63-Gly 64, which acts as a green chromophore that is photoconvertible to red following (ultra-) violet irradiation. Here, we report the molecular cloning and crystal structure determination of a new fluorescent protein, KikG, from the coral Favia favus, and its in vitro evolution conferring green-to-red photoconvertibility. Substitution of the His 62-Tyr 63-Gly 64 sequence into the native protein provided only negligible photoconversion. On the basis of the crystal structure, semi-rational mutagenesis of the amino acids surrounding the chromophore was performed, leading to the generation of an efficient highlighter, KikGR. Within mammalian cells, KikGR is more efficiently photoconverted and is several-fold brighter in both the green and red states than Kaede. In addition, KikGR was successfully photoconverted using two-photon excitation microscopy at 760 nm, ensuring optical cell labelling with better spatial discrimination in thick and highly scattering tissues.

SUBMITTER: Tsutsui H

PROVIDER: S-EPMC1299271 | biostudies-literature | 2005 Mar

REPOSITORIES: biostudies-literature

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Semi-rational engineering of a coral fluorescent protein into an efficient highlighter.

Tsutsui Hidekazu H Karasawa Satoshi S Shimizu Hideaki H Nukina Nobuyuki N Miyawaki Atsushi A

EMBO reports 20050301 3

Kaede is a natural photoconvertible fluorescent protein found in the coral Trachyphyllia geoffroyi. It contains a tripeptide, His 62-Tyr 63-Gly 64, which acts as a green chromophore that is photoconvertible to red following (ultra-) violet irradiation. Here, we report the molecular cloning and crystal structure determination of a new fluorescent protein, KikG, from the coral Favia favus, and its in vitro evolution conferring green-to-red photoconvertibility. Substitution of the His 62-Tyr 63-Gly ...[more]

PMID: 15731765

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Project description:BackgroundThe ability of fungal cellobiose dehydrogenase (CDH) to generate H2O2 in-situ is highly interesting for biotechnological applications like cotton bleaching, laundry detergents or antimicrobial functionalization of medical devices. CDH's ability to directly use polysaccharide derived mono- and oligosaccharides as substrates is a considerable advantage compared to other oxidases such as glucose oxidase which are limited to monosaccharides. However CDH's low activity with oxygen as electron acceptor hampers its industrial use for H2O2 production. A CDH variant with increased oxygen reactivity is therefore of high importance for biotechnological application. Uniform expression levels and an easy to use screening assay is a necessity to facilitate screening for CDH variants with increased oxygen turnover.ResultsA uniform production and secretion of active Myriococcum thermophilum CDH was obtained by using Saccharomyces cerevisiae as expression host. It was found that the native secretory leader sequence of the cdh gene gives a 3 times higher expression than the prepro leader of the yeast α-mating factor. The homogeneity of the expression in 96-well deep-well plates was good (variation coefficient <15%). A high-throughput screening assay was developed to explore saturation mutagenesis libraries of cdh for improved H2O2 production. A 4.5-fold increase for variant N700S over the parent enzyme was found. For production, N700S was expressed in P. pastoris and purified to homogeneity. Characterization revealed that not only the kcat for oxygen turnover was increased in N700S (4.5-fold), but also substrate turnover. A 3-fold increase of the kcat for cellobiose with alternative electron acceptors indicates that mutation N700S influences the oxidative- and reductive FAD half-reaction.ConclusionsSite-directed mutagenesis and directed evolution of CDH is simplified by the use of S. cerevisiae instead of the high-yield-host P. pastoris due to easier handling and higher transformation efficiencies with autonomous plasmids. Twelve clones which exhibited an increased H2O2 production in the subsequent screening were all found to carry the same amino acid exchange in the cdh gene (N700S). The sensitive location of the five targeted amino acid positions in the active site of CDH explains the high rate of variants with decreased or entirely abolished activity. The discovery of only one beneficial exchange indicates that a dehydrogenase's oxygen turnover is a complex phenomenon and the increase therefore not an easy target for protein engineering.

Dataset Information

Semi-rational engineering of a coral fluorescent protein into an efficient highlighter.

Publications

Semi-rational engineering of a coral fluorescent protein into an efficient highlighter.

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