Use of Megaprimer and Overlapping Extension PCR (OE-PCR) to Mutagenize and Enhance Cyclodextrin Glucosyltransferase (CGTase) Function.
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.
['Goh|Kian Mau|KM|', 'Liew|Kok Jun|KJ|', 'Chai|Kian Piaw|KP|', 'Illias|Rosli Md|RM|']
D000595:Amino Acid Sequence D001407:Bacillus D001483:Base Sequence D001665:Binding Sites D003460:Crystallization D018360:Crystallography, X-Ray D003505:Cyclodextrins D017931:DNA Primers D005964:Glucosyltransferases D016297:Mutagenesis, Site-Directed D009153:Mutagens D016133:Polymerase Chain Reaction D015202:Protein Engineering
Overlapping extension PCR CGTase Protein engineering Megaprimer PCR Bacillus Rational design