Analysis of shuffled gene libraries

Joern, John M.; Meinhold, Peter; Arnold, Frances H.

doi:10.1006/jmbi.2001.5349

Cited by 87 publications

(61 citation statements)

References 30 publications

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“…Classical DNA shuffling was not feasible, as the identity between VEGF and VEGF-C is concentrated within the cystine knot; therefore, this method would have caused a strong crossover bias (30). A non-random approach resulted in a library size that allowed exhaustive profiling of the receptor binding pattern of each individual clone.…”

Section: Discussionmentioning

confidence: 99%

Vascular Endothelial Growth Factor (VEGF)/VEGF-C Mosaic Molecules Reveal Specificity Determinants and Feature Novel Receptor Binding Patterns

Jeltsch

Kärpänen

Strandin

et al. 2006

Journal of Biological Chemistry

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Section: Discussionmentioning

confidence: 99%

Vascular Endothelial Growth Factor (VEGF)/VEGF-C Mosaic Molecules Reveal Specificity Determinants and Feature Novel Receptor Binding Patterns

Jeltsch

Kärpänen

Strandin

et al. 2006

Journal of Biological Chemistry

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“…Subsequently, Saraf and Maranas (14) proposed a rapid method to identify directly such clashes between contacting residue pairs in the protein hybrids. Comparison with sequence data of functional clones derived from many studies (4,11,(15)(16)(17) revealed that the method was capable of classifying hybrids (crossover combinations) as functional or nonfunctional accounting for mirror chimeras. However, neither this method nor any of those discussed earlier manage to a priori rank functional hybrids with respect to their level of activity.…”

mentioning

confidence: 99%

FamClash: A method for ranking the activity of engineered enzymes

Saraf

Horswill

Benkovic

et al. 2004

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

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This article introduces the computational procedure FamClash for analyzing incompatibilities in engineered protein hybrids by using protein family sequence data. All pairs of residue positions in the sequence alignment that conserve the property triplet of charge, volume, and hydrophobicity are first identified, and significant deviations are denoted as residue-residue clashes. This approach moves beyond earlier efforts aimed at solely classifying hybrids as functional or nonfunctional by correlating the rank ordering of these hybrids based on their activity levels. Experimental testing of this approach was performed in parallel to assess the predictive ability of FamClash. As a model system, single-crossover ITCHY (incremental truncation for the creation of hybrid enzymes) libraries were prepared from the Escherichia coli and Bacillus subtilis dihydrofolate reductases, and the activities of functional hybrids were determined. Comparisons of the predicted clash map as a function of crossover position revealed good agreement with activity data, reproducing the observed V shape and matching the location of a local peak in activity.protein engineering ͉ dihydrofolate reductase ͉ residue-residue clash ͉ computational hybrid prescreening ͉ incremental truncation R ecent advances in protein engineering (1-5) have allowed researchers to go beyond the limitations of homologydependent directed evolution methods. The ability to freely explore protein sequence space has revealed a number of troublesome trends. First, the lower the sequence identity of the recombined parental sequences, the smaller the percentage of the combinatorial protein library that remains functional (2, 4). This has been reported in several studies (6-8) using differing protocols, thus implicating the global nature of this effect. More troublesome is the finding that the remaining functional hybrids tend to have only residual activities. Therefore, it appears that exploring protein sequence space freely comes at the expense of severely degrading the average stability and functionality of the combinatorial library. This has motivated the development of computational methods to prescreen hybrids for their potential of being stably folded (9) and functional. These analyses then serve to direct the sampling of protein sequences by the combinatorial library toward desirable regions in sequence space. Specifically, favorable positions for junctions between fragments from different parental sequences can be identified, and restrictions can be imposed on sets of parental sequences that contribute fragments to a particular junction.Therefore, further improvements in the stability and functionality of hybrid proteins may be attained by developing quantitative methods that identify deleterious interactions arising from residue pairs within the gene fragment combinations. To this end, Monte Carlo simulations by Bogarad and Deem (10) suggested that swapping of low-energy structures is least disruptive to protein structure. The SCHEMA algorithm (11) postulates t...

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“…The application of family gene shuffling strategy has been demonstrated by the previous reports (Crameri et al 1998;Niederhauser et al 2012;Madan and Mishra 2014). It was indicated that the gene sequence homology of parent template should be more than 70% for gene shuffling in order to achieve the favorable result (Joern et al 2002). Although the sequence homology of four protease genes in this study was only 42%, the good result was achieved by increasing PCR cycles from 15 to 40 without primers, indicating that the higher homology was not essential for DNA shuffling.…”

Section: Discussionmentioning

confidence: 60%