Abstract
Directed evolution is an often used approach toward new proteins with tailor-made properties. It consists of random variation of the coding sequence of a protein followed by an appropriate selection procedure or a suitable type of property read out. In many, if not all cases, it is of significant advantage to constrain the randomly mutagenized DNA sequence to that encoding a particular part of the protein or a distinct domain, and not to mutagenize the entire gene of the target protein. For this purpose, a three-step, polymerase-based method was developed, which is independent of two flanking restriction sites adjacent to the nucleotide sequence supposed to be mutagenized, and named protein library generation by overlap extension (PDLGO).
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References
Arnold F.H. (1998) Design by directed evolution. Acc. Chem. Res. 31, 125–131.
Fasan R., Chen, M.M., Crook, N.C., Arnold, F.H. (2007) Engineered alkane-hydroxylating cytochrome P450BM3 exhibiting nativelike catalytic properties. Angew. Chem. Int. Ed. Engl. 46, 8414–8418.
Leung D., Chen, E., Goeddel, D. (1989) A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1, 11–15.
Talker-Huiber D., Jose, J., Glieder, A., Pressnig, M., Stubenrauch, G., Schwab, H. (2003) Esterase EstE from Xanthomonas vesicatoria (Xv_EstE) is an outer membrane protein capable of hydrolyzing long-chain polar esters. Appl. Microbiol. Biotechnol. 61, 479–487.
Horton R.M., Hunt, H.D., Ho, S.N., Pullen, J.K., Pease, L.R. (1989) Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–68.
Heckman K.L., Pease, L.R. (2007) Gene splicing and mutagenesis by PCR-driven overlap extension. Nat. Protoc. 2, 924–932.
Gratz A., Jose, J. (2008) Protein domain library generation by overlap extension (PDLGO): a tool for enzyme engineering. Anal. Biochem. 378, 171–176.
Vega J., Puebla, C., Vasquez, R., Farias, M., Alarcon, J., Pastor-Anglada, M., Krause, B., Casanello, P., Sobrevia, L. (2009) TGF-{beta}1 inhibits expression and activity of hENT1 in a nitric oxide-dependent manner in human umbilical vein endothelium. Cardiovasc. Res. 82, 458–467.
Bastian S., Rekowski, M.J., Witte, K., Heckmann-Pohl, D.M., Giffhorn, F. (2005) Engineering of pyranose 2-oxidase from Peniophora gigantea towards improved thermostability and catalytic efficiency. Appl. Microbiol. Biotechnol. 67, 654–663.
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Gratz, A., Jose, J. (2011). Focusing Mutations Within Random Libraries to Distinct Areas: Protein Domain Library Generation by Overlap Extension. In: Lu, C., Browse, J., Wallis, J. (eds) cDNA Libraries. Methods in Molecular Biology, vol 729. Humana Press. https://doi.org/10.1007/978-1-61779-065-2_10
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DOI: https://doi.org/10.1007/978-1-61779-065-2_10
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