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PubMed · 12206782

Structure-based combinatorial protein engineering (SCOPE).

Paul E O'Maille·Marina Bakhtina·Ming-Daw Tsai
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Abstract

Presented here is the development a semi-rational protein engineering approach that uses information from protein structure coupled with established DNA manipulation techniques to design and create multiple crossover libraries from non-homologous genes. The utility of structure-based combinatorial protein engineering (SCOPE) was demonstrated by its application to two distantly related members of the X-family of DNA polymerases: rat DNA polymerase beta (Pol beta) and African swine fever virus DNA polymerase X (Pol X). These proteins share similar folds but have low sequence identity, and differ greatly in both size and activity. "Equivalent" subdomain elements of structure were designed on the basis of the tertiary structure of Pol beta and the corresponding regions of Pol X were inferred from homology modeling and sequence alignment analysis. Libraries of chimeric genes with up to five crossovers were synthesized in a series of PCR reactions by employing hybrid oligonucleotides that code for variable connections between structural elements. Genetic complementation in Escherichia coli enabled identification of several novel DNA polymerases with enhanced phenotypes. Both the composition of structural elements and the manner in which they were linked were shown to be essential for this property, indicating the importance of these aspects of design.

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African Swine Fever VirusAmino Acid SequenceAnimalsDNA Polymerase betaDNA-Directed DNA PolymeraseEscherichia coliGenetic Complementation TestMolecular Sequence DataPeptide LibraryPhenotypePolymerase Chain ReactionProtein EngineeringProtein Structure, TertiaryRatsRecombinant Fusion ProteinsSequence AlignmentTransformation, BacterialJournal ArticleResearch Support, U.S. Gov't, P.H.S.

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Paul E O'Maille, Marina Bakhtina, Ming-Daw Tsai. 2002-08-23. Structure-based combinatorial protein engineering (SCOPE).. https://doi.org/10.1016/s0022-2836(02)00675-7

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