Growth factor engineering by degenerate homoduplex gene family recombination

Coco, Wayne M.; Encell, Lance P.; Levinson, William E.; Crist, Michael; Loomis, A. Katrina; Licato, Laura L.; Arensdorf, Joseph J.; Sica, Nicole; Pienkos, Philip T.; Monticello, Daniel J.

doi:10.1038/nbt757

Cited by 71 publications

(34 citation statements)

References 23 publications

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“…Designed libraries can be synthesized for roughly the same cost as a designed sequence by recognizing the opportunities in gene synthesis for the combinatorial shuffling of sequence diversity (14)(15)(16)(17). Although many algorithms have now been proposed to design such combinatorial libraries (7-9, 11, 12), few computationally designed libraries have been characterized experimentally (9,18,19), and, to our knowledge, there have been no controlled experiments comparing these methods with each other or with libraries of randomly generated sequence diversity.…”

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confidence: 99%

Computationally designed libraries of fluorescent proteins evaluated by preservation and diversity of function

Treynor

Vizcarra²,

Nedelcu

et al. 2007

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

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To determine which of seven library design algorithms best introduces new protein function without destroying it altogether, seven combinatorial libraries of green fluorescent protein variants were designed and synthesized. Each was evaluated by distributions of emission intensity and color compiled from measurements made in vivo. Additional comparisons were made with a library constructed by error-prone PCR. Among the designed libraries, fluorescent function was preserved for the greatest fraction of samples in a library designed by using a structure-based computational method developed and described here. A trend was observed toward greater diversity of color in designed libraries that better preserved fluorescence. Contrary to trends observed among libraries constructed by error-prone PCR, preservation of function was observed to increase with a library's average mutation level among the four libraries designed with structure-based computational methods.GFP ͉ library design ͉ protein design ͉ protein engineering ͉ high-throughput screening P rotein sequence space is so vast that one can easily imagine the optimal sequence for a particular application will never be sampled by random mutation and recombination. Structure-based computational protein design tools seek to screen that sequence space more thoroughly than can be screened in the laboratory, but are currently based on approximate representations of candidate sequences and an incomplete understanding of the relationships between structure and function. Although many algorithms used to screen sequences in silico aim to identify a single optimal sequence (1-5), others aim instead to optimize the composition of a library of sequences (6-13). Provided that resources exist to synthesize and screen such libraries, library design algorithms compensate for the approximations built into them by increasing the number of attempts at designing the desired function. Viewed from a complementary perspective, such algorithms aim to sample sequence space more effectively than methods that randomly generate sequence diversity.Designed libraries can be synthesized for roughly the same cost as a designed sequence by recognizing the opportunities in gene synthesis for the combinatorial shuffling of sequence diversity (14-17). Although many algorithms have now been proposed to design such combinatorial libraries (7-9, 11, 12), few computationally designed libraries have been characterized experimentally (9,18,19), and, to our knowledge, there have been no controlled experiments comparing these methods with each other or with libraries of randomly generated sequence diversity. The results of such a comparison would be hard to predict, especially because none of these methods models protein function explicitly. Instead, these algorithms attempt to model protein stability as a surrogate for protein function on the assumption that libraries with a greater fraction of well folded proteins are more likely to contain variants with the desired function.Here, we evaluate seven design...

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confidence: 99%

Computationally designed libraries of fluorescent proteins evaluated by preservation and diversity of function

Treynor

Vizcarra²,

Nedelcu

et al. 2007

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

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“…We randomized specificity-determining residues that were identified in our previous study (Ser 557 , Asn 566 , Lys 568 ) by saturation mutagenesis (also called combinatorial cassette mutagenesis, Refs. [22][23][24][25][26][27]. This strategy effected vast improvements in a single round of directed evolution.…”

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confidence: 99%

Rapid Evolution of β-Glucuronidase Specificity by Saturation Mutagenesis of an Active Site Loop

Geddie

Matsumura

2004

Journal of Biological Chemistry

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Protein engineers have widely adopted directed evolution as a design algorithm, but practitioners have not come to a consensus about the best method to evolve protein molecular recognition. We previously used DNA shuffling to direct the evolution of Escherichia coli ␤-glucuronidase (GUS) variants with increased ␤-galactosidase activity. Epistatic (synergistic) mutations in amino acids 557, 566, and 568, which are part of an active site loop, were identified in that experiment (Matsumura, I., and Ellington, A. D. (2001) J. Mol. Biol. 305, 331-339). Here we show that site saturation mutagenesis of these residues, overexpression of the resulting library in E. coli, and high throughput screening led to the rapid evolution of clones exhibiting increased activity in reactions with p-nitrophenyl-␤-D-xylopyranoside (pNP-xyl). The xylosidase activities of the 14 fittest clones were 30-fold higher on average than that of the wild-type GUS. The 14 corresponding plasmids were pooled, amplified by long PCR, self-ligated with T4 DNA ligase, and transformed into E. coli. Thirteen clones exhibiting an average of 80-fold improvement in xylosidase activity were isolated in a second round of screening. One of the evolved proteins exhibited a ϳ200-fold improvement over the wild type in reactivity (k cat /K m ) with pNP-xyl, with a 290,000-fold inversion of specificity. Sequence analysis of the 13 round 2 isolates suggested that all were products of intermolecular recombination events that occurred during whole plasmid PCR. Further rounds of evolution using DNA shuffling and staggered extension process (StEP) resulted in modest improvement. These results underscore the importance of epistatic interactions and demonstrate that they can be optimized through variations of the facile whole plasmid PCR technique.

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“…In the case of class IIa bacteriocins, however, the parent peptides are so short in length that it was feared that DNase I digestion might yield fragments which could be too small to effectively ligate in subsequent steps. Recently, a synthetic PCR-based method not using DNase I was reported for DNA shuffling of growth factors and subtilisin (11,36), which used synthetic oligonucleotides as fragmented DNA and linked them by annealing the homologous scaffold and subsequently performing PCR. Thus, the synthetic method was applied for the class IIa bacteriocins, with modifications to allow DNA shuffling by sequential PCR extension reactions, since the bacteriocins were too short to anneal the homologous region adequately in comparison with the annealing achieved in the previous studies.…”

Section: Discussionmentioning

confidence: 99%

Development of Innovative Pediocin PA-1 by DNA Shuffling among Class IIa Bacteriocins

Tominaga

Hatakeyama

2007

Appl Environ Microbiol

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Pediocin PA-1 is a member of the class IIa bacteriocins, which show antimicrobial effects against lactic acid bacteria. To develop an improved version of pediocin PA-1, reciprocal chimeras between pediocin PA-1 and enterocin A, another class IIa bacteriocin, were constructed. Chimera EP, which consisted of the C-terminal half of pediocin PA-1 fused to the N-terminal half of enterocin A, showed increased activity against a strain of Leuconostoc lactis isolated from a sour-spoiled dairy product. To develop an even more effective version of this chimera, a DNA-shuffling library was constructed, wherein four specific regions within the N-terminal half of pediocin PA-1 were shuffled with the corresponding sequences from 10 other class IIa bacteriocins. Activity screening indicated that 63 out of 280 shuffled mutants had antimicrobial activity. A colony overlay activity assay showed that one of the mutants (designated B1) produced a >7.8-mm growth inhibition circle on L. lactis, whereas the parent pediocin PA-1 did not produce any circle. Furthermore, the active shuffled mutants showed increased activity against various species of Lactobacillus, Pediococcus, and Carnobacterium. Sequence analysis revealed that the active mutants had novel N-terminal sequences; in active mutant B1, for example, the parental pediocin PA-1 sequence (KYYGNGVTCGKHSC) was changed to TKYYGNGVSCTKSGC. These new and improved DNA-shuffled bacteriocins could prove useful as food additives for inhibiting sour spoilage of dairy products.

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Growth factor engineering by degenerate homoduplex gene family recombination

Cited by 71 publications

References 23 publications

Computationally designed libraries of fluorescent proteins evaluated by preservation and diversity of function

Computationally designed libraries of fluorescent proteins evaluated by preservation and diversity of function

Rapid Evolution of β-Glucuronidase Specificity by Saturation Mutagenesis of an Active Site Loop

Development of Innovative Pediocin PA-1 by DNA Shuffling among Class IIa Bacteriocins

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