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Methods Mol Biol. 2017;1498:385-396.

Use of Megaprimer and Overlapping Extension PCR (OE-PCR) to Mutagenize and Enhance Cyclodextrin Glucosyltransferase (CGTase) Function.

Author information

1
Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia. gohkianmau@utm.my.
2
Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.
3
Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.

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.

KEYWORDS:

Bacillus; CGTase; Megaprimer PCR; Overlapping extension PCR; Protein engineering; Rational design

PMID:
27709591
DOI:
10.1007/978-1-4939-6472-7_27
[Indexed for MEDLINE]

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