Use of megaprimer and overlapping extension pcr (OE-PCR) to mutagenize and enhance cyclodextrin glucosyltransferase (CGTASE) function

• Main Authors: Goh, Kian Mau, Liew, Kok Jun, Chai, Kian Piaw, Md. Illias, Rosli
• Format: Book Section
• Published: Humana Press Inc. 2017
• Subjects: TP Chemical technology
• Online Access: http://eprints.utm.my/id/eprint/97041/; http://dx.doi.org/10.1007/978-1-4939-6472-7_27

Protein engineering is a very useful tool for probing structure–function relationships in proteins. Specifically, site-directed mutagenized proteins can provide useful insights into structural, binding and catalytic mechanisms of a protein, particularly when coupled with crystallization. In this chapter, we describe two protocols for performing site-directed mutagenesis of any protein-coding sequence, namely, megaprimer PCR and overlapping extension PCR (OE-PCR). We use as an example how these two SDM methods enhanced the function of a cyclodextrin glucosyltransferase (CGTase) from Bacillus lehensis strain G1.Bacillus, CGTase, Megaprimer PCR, Overlapping extension PCR, Protein engineering, Rational designProtein engineering is a very useful tool for probing structure–function relationships in proteins. Specifically, site-directed mutagenized proteins can provide useful insights into structural, binding and catalytic mechanisms of a protein, particularly when coupled with crystallization. In this chapter, we describe two protocols for performing site-directed mutagenesis of any protein-coding sequence, namely, megaprimer PCR and overlapping extension PCR (OE-PCR). We use as an example how these two SDM methods enhanced the function of a cyclodextrin glucosyltransferase (CGTase) from Bacillus lehensis strain G1.