GFPs of insertion mutation generated by molecular size‐altering block shuffling

Kitamura, Koichiro; Yoshida, Chuya; Nishigaki, Koichi

doi:10.1016/s0014-5793(03)01308-5

Cited by 5 publications

(6 citation statements)

References 20 publications

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“…This result agrees with those found by Li et al [10] and Kitamura et al [11]. Li removed the region comprising amino acids 132–139 of GFP by site-directed mutagenesis, whereas Kitamura randomly removed tri-peptide blocks in the region 125–142.…”

Section: Resultssupporting

confidence: 92%

“…We selected the region located between residues 129–142 as target of the mutagenesis for three reasons: 1) It is the longest loop of the protein; 2) two previous attempts of deletions in this area failed to produce fluorescent proteins [10,11]; 3) Published sequence alignments of GFP versus GFP-like proteins of anthozoas suggest that GFP may tolerate deletion of either G138 [16] or H139 [17] (G139 and H140 respectively in sgGFP).…”

Section: Resultsmentioning

confidence: 99%

“…GFP tolerates enlargement through the insertion of short peptides [11], long peptides [12] and even complete proteins [13] in different locations, but it is particularly sensitive to shortening by internal site-directed deletions [10,11]. Prior to this study, only amino- or carboxyl-terminus deletions have been reported for GFP [10].…”

Section: Introductionmentioning

confidence: 99%

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The effect of amino acid deletions and substitutions in the longest loop of GFP

et al. 2007

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BackgroundThe effect of single and multiple amino acid substitutions in the green fluorescent protein (GFP) from Aequorea victoria has been extensively explored, yielding several proteins of diverse spectral properties. However, the role of amino acid deletions in this protein -as with most proteins- is still unknown, due to the technical difficulties involved in generating combinatorial in-phase amino acid deletions on a target region.ResultsIn this study, the region I129-L142 of superglo GFP (sgGFP), corresponding to the longest loop of the protein and located far away from the central chromophore, was subjected to a random amino acid deletion approach, employing an in-house recently developed mutagenesis method termed Codon-Based Random Deletion (COBARDE). Only two mutants out of 16384 possible variant proteins retained fluorescence: sgGFP-Δ I129 and sgGFP-Δ D130. Interestingly, both mutants were thermosensitive and at 30°C sgGFP-Δ D130 was more fluorescent than the parent protein. In contrast with deletions, substitutions of single amino acids from residues F131 to L142 were well tolerated. The substitution analysis revealed a particular importance of residues F131, G135, I137, L138, H140 and L142 for the stability of the protein.ConclusionThe behavior of GFP variants with both amino acid deletions and substitutions demonstrate that this loop is playing an important structural role in GFP folding. Some of the amino acids which tolerated any substitution but no deletion are simply acting as "spacers" to localize important residues in the protein structure.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

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The effect of amino acid deletions and substitutions in the longest loop of GFP

et al. 2007

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“…While many methods improve functions through creating and recombining point mutations, Y-ligation-based block shuffling (YLBS) is a general methodology that mimics evolution processes such as domain shuffling, exon shuffling, and module shuffling, and it can be used for generating high-diversity libraries (155,156). YLBS is based on repeated cycles of ligation of sequence blocks with a stem and two branches (Y-ligation) formed by two types of single-stranded DNA.…”

Section: Y-ligation-based Block Shufflingmentioning

confidence: 99%

Laboratory-Directed Protein Evolution

Yuan

Kurek

English

et al. 2005

Microbiol Mol Biol Rev

175

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Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to “evolve” in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences

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“…I digestion as described by Stemmer (1994), but by using overlapping DNA blocks and thus corresponds to a PCR fusion technique as described by Shevchuk et al (2004) with DNA blocks of around 100-300 bp leading to a full-length sequence of 800 bp. This use of blocks has been explored before in order to engineer novel proteins from amino acid blocks (Kitamura et al, 2003;Shiba et al, 2002). Our metagenomic approach used the shuffling process not only to examine possible genetic diversity generated during the protein engineering (Stemmer, 1994) but also to explore the metagenome DNA diversity independent of bacterial culture by including it directly in the construction of this gene (Fig.…”

Section: Metagenomic Shufflingmentioning

confidence: 99%