Combinatorial protein engineering by incremental truncation

Ostermeier, Marc; Nixon, Andrew E.; Shim, Jae Hoon; Benkovic, Stephen J.

doi:10.1073/pnas.96.7.3562

Cited by 125 publications

(108 citation statements)

References 30 publications

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“…Earlier work has shown that folded hybrid domains can be created from homologous proteins (18)(19)(20). With our hybrid domains we were unable to detect any homologies between CspA and any of the donor proteins by sequence comparisons alone.…”

Section: Resultsmentioning

confidence: 75%

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Novel folded protein domains generated by combinatorial shuffling of polypeptide segments

Riechmann

Winter

2000

Proc. Natl. Acad. Sci. U.S.A.

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It has been proposed that the architecture of protein domains has evolved by the combinatorial assembly and͞or exchange of smaller polypeptide segments. To investigate this proposal, we fused DNA encoding the N-terminal half of a ␤-barrel domain (from cold shock protein CspA) with fragmented genomic Escherichia coli DNA and cloned the repertoire of chimeric polypeptides for display on filamentous bacteriophage. Phage displaying folded polypeptides were selected by proteolysis; in most cases the proteaseresistant chimeric polypeptides comprised genomic segments in their natural reading frames. Although the genomic segments appeared to have no sequence homologies with CspA, one of the originating proteins had the same fold as CspA, but another had a different fold. Four of the chimeric proteins were expressed as soluble polypeptides; they formed monomers and exhibited cooperative unfolding. Indeed, one of the chimeric proteins contained a set of very slowly exchanging amides and proved more stable than CspA itself. These results indicate that native-like proteins can be generated directly by combinatorial segment assembly from nonhomologous proteins, with implications for theories of the evolution of new protein folds, as well as providing a means of creating novel domains and architectures in vitro.

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Section: Resultsmentioning

confidence: 75%

“…Experimentally, it has been shown that DNA shuffling of homologous genes can generate folded domains with improved properties (18)(19)(20). However, it is unlikely that such homologous recombinations can lead to the creation of entirely new protein folds, which are more likely to require the recombination of nonhomologous genes (3).…”

mentioning

confidence: 99%

Novel folded protein domains generated by combinatorial shuffling of polypeptide segments

Riechmann

Winter

2000

Proc. Natl. Acad. Sci. U.S.A.

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“…Exchange of the C-terminal helix of the rat enzyme with the corresponding sequence from the human protein in clones RH-A5 and RH-F2 produced proteins that displayed 30-and 10-fold rate enhancements, respectively, relative to hGSTT1-1 but were nonetheless substantially less active than rGSTT2-2. Such reductions in catalytic rate relative to the most active parent are typical of chimeric enzymes derived from homologyindependent recombination (15,17,29).…”

Section: Resultsmentioning

confidence: 99%

“…Recently, several techniques for the combinatorial generation of protein chimeras in regions of low homology have been developed. These techniques include nonhomologous random recombination (15), sequence homologyindependent protein recombination (16), and incremental truncation for the creation of hybrid enzymes (ITCHY) and SCRATCHY (17,18). However, random nonhomologous recombination results in the creation of libraries containing a large number of out-of-frame or otherwise inactive clones.…”

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confidence: 99%

Evolution of highly active enzymes by homology-independent recombination

Griswold

Kawarasaki

Ghoneim

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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The theta-class GST enzymes hGSTT1-1 (human GST -1-1) and rGSTT2-2 (rat GST -2-2) share 54.3% amino acid identity and exhibit different substrate specificities. Homology-independent techniques [incremental truncation for the creation of hybrid enzymes (ITCHY) and SCRATCHY] and low-homology techniques (recombination-dependent exponential amplification PCR) were used to create libraries of chimeric enzymes containing crossovers (C͞Os) at positions not accessible by DNA family shuffling. Highthroughput flow cytometric screening using the fluorogenic rGSTT2-2-specific substrate 7-amino-4-chloromethyl coumarin led to the isolation of active variants with either one or two C͞Os. One of these enzymes, SCR23 (83% identity to hGSTT1-1), was encoded by a gene that exchanged helices 4 and 5 of hGSTT1-1 with the corresponding sequence from rGSTT2-2. Compared with either parent, this variant was found to have an improved k cat with the selection substrate and also exhibited activity for the conjugation of glutathione to ethacrynic acid, a compound that is not recognized by either parental enzyme. These results highlight the power of combinatorial homology-independent and lowhomology recombination methods for the generation of unique, highly active enzymes and also suggest a possible means of enzyme ''humanization.'' enzyme engineering ͉ enzyme humanization ͉ GST ͉ ITCHY ͉ high-throughput screening G STs play a crucial role in cellular detoxification by conjugating reactive electrophilic compounds to the tripeptide glutathione (GSH) (1). Mammals encode at least seven distinct classes of GSTs. The theta enzymes represent the oldest class of GSTs and, as such, are produced by a surprisingly diverse array of animals, plants, algae, and bacteria (2). Although theta-class enzymes retain the highly conserved GST 3D fold, they possess a characteristic C-terminal ␣-helical extension, which, unlike that of some alpha-class enzymes, covers both the electrophile and GSH-binding sites, thus clearly differentiating the theta class from other members of the soluble GST superfamily (3).The rat GST -2-2 (rGSTT2-2, 244 aa) and human GST -1-1 (hGSTT1-1, 240 aa) enzymes exhibit 54.3% overall amino acid identity. The GSH-binding domain, or G site, of the two enzymes (residues Ϸ1-77) is conserved (79.2% amino acid identity), and the 5Ј regions that encode this domain (nucleotides 1-231) exhibit 74.5% DNA sequence identity. In contrast, the electrophilic substrate binding domain, or H site (amino acid Ϸ89 to termini), of the rat and human enzymes (encoded by nucleotides Ϸ265-732 in rGSTT2-2 and nucleotides Ϸ265-720 in hG-STT1-1) exhibit only 41.4% amino acid identity and 57.9% DNA sequence identity. The sequence divergence of the two enzymes in the C-terminal domain is manifest in their respective electrophilic substrate selectivities. The rat enzyme preferentially catalyzes the GSH conjugation of 1-menaphythyl sulfate, whereas hGSTT1-1 exhibits a characteristic reactivity with dichloromethane (2). Additionally, we recently showed that rG-STT2-2 e...

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“…Artificial splitting of membrane proteins, which can form a catalytically active heterodimer complex, have been described earlier, [23][24][25] but such a conversion is not feasible in some cases, presumably due to insufficient assembly or improper folding of fragments. 26) Several split proteins were active only in the case of coexpression of the polypeptides in a restricted space in the cell, from a dicistronic operon on a single plasmid for example. 24) We also examined the effect of separate expression of the lysE24-N and lysE24-C regions from two plasmids in M. methylotrophus, and observed no enhancement of L-lysine production.…”

Section: Discussionmentioning

confidence: 99%