Structure-based combinatorial protein engineering (SCOPE).
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.
O'Maille
Paul E
PE
Ohio State Biochemistry Program, Ohio State University, 100 West 18th Avenue, Columbus, OH 43210-1173, USA.
Bakhtina
Marina
M
Tsai
Ming-Daw
MD
D000358:African Swine Fever Virus / Q000201:enzymology / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D019151:Peptide Library; D010641:Phenotype; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014169:Transformation, Bacterial
D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C109534:DNA polymerase X; D019951:DNA Polymerase beta; D004259:DNA-Directed DNA Polymerase