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.
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Acknowledgments
This work was financially supported by Universiti Teknologi Malaysia (GUP 09H98 and 06H31).
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Goh, K.M., Liew, K.J., Chai, K.P., Illias, R.M. (2017). Use of Megaprimer and Overlapping Extension PCR (OE-PCR) to Mutagenize and Enhance Cyclodextrin Glucosyltransferase (CGTase) Function. In: Reeves, A. (eds) In Vitro Mutagenesis. Methods in Molecular Biology, vol 1498. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6472-7_27
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DOI: https://doi.org/10.1007/978-1-4939-6472-7_27
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6470-3
Online ISBN: 978-1-4939-6472-7
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