Site-saturation Mutagenesis Library Construction to characterize structure-function relationships (CAT#: STEM-MB-0023-WXH)

Introduction

Site-saturation mutagenesis (SSM) is a simple, semi-rational approach to protein engineering that occupies a useful middle ground between fully rational site-directed mutagenesis strategies and non-rational mutagenesis strategies. In SSM, the semi-rational designation derives from the fact that target residues are identified in a specific, knowledge-guided manner, but are then mutated randomly, in either an individual or combinatorial fashion. Through the site-saturation mutagenesis, it is possible to create a library of mutants containing all possible combinations of 20 different amino acids at one or more predetermined positions in a gene sequence. SSM is a powerful approach in protein engineering to characterize structure-function relationships, as well as to create improved protein variants.

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Principle Applications Procedure Materials Notes Related Products

Applications

• Generate novel protein variants
• Directed evolution
• Structure–function studies
• Fine-tune enzyme properties including substrate specificity, thermostability or simply to increase activity

Procedure

1.Prepare the template plasmid DNA
2.Harvest bacterial culture for plasmid isolation
3.Lyse the bacterial culture
4.Design forward and reverse primers
5.Run PCR-based SSM
6.Analyze the PCR product by gel electrophoresis
7.Digest the template DNA with DpnI
8.Transform the plasmid into E. coli

Notes

Information to be provided by customers:
• name of cloning vector and cloning site
• the Latin name of the host cell.
• the amino acid sequence containing the mutation site or the gene sequence of an existing template and the location where the mutation is required.

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