title: Use of Megaprimer and Overlapping Extension PCR (OE-PCR) to Mutagenize and Enhance Cyclodextrin Glucosyltransferase (CGTase) Function.
abstract: 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.
authors.affiliation: Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia. gohkianmau@utm.my.
authors.forename: Kian Mau
authors.initials: KM
authors.lastname: Goh
authors.affiliation: Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.
authors.forename: Kok Jun
authors.initials: KJ
authors.lastname: Liew
authors.affiliation: Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.
authors.forename: Kian Piaw
authors.initials: KP
authors.lastname: Chai
authors.affiliation: Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.
authors.forename: Rosli Md
authors.initials: RM
authors.lastname: Illias
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray / Q000379:methods; D003505:Cyclodextrins / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics*; D005964:Glucosyltransferases / Q000235:genetics*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009153:Mutagens / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D015202:Protein Engineering / Q000379:methods
chemical_list: D003505:Cyclodextrins; D017931:DNA Primers; D009153:Mutagens; D005964:Glucosyltransferases
keywords: Bacillus; CGTase; Megaprimer PCR; Overlapping extension PCR; Protein engineering; Rational design