Codon-Based Mutagenesis - Introduction

Introduction to Codon-Based Mutagenesis

Directed molecular evolution and combinatorial methods are key strategies used for protein engineering research. These approaches commonly involves using partially randomized synthetic oligonucleotides to generate a partially randomized gene library, expressing it in an appropriate vector to generate the protein set encoded by the library, and then screening the expressed proteins for improved or modified characteristics. Examples of the latter include changes in protein-ligand binding affinity, enzyme selectivity, or protein stability. While a variety of site-directed mutagenesis methods can be employed to introduce randomization at specific base positions of a gene, such methods often introduce significant amounts of undesirable codon bias into the library, due to the mismatch between the base-by-base nature of classical oligonucleotide synthesis and the triplet nature of the codon (1). The problem of unwanted codon bias can be eliminated by synthesizing the random portion of the oligos codon-by-codon, with trimer phosphoramidites, instead of base-by-base. This codon-based mutagenesis system conveys the ability to effectively control codon bias in the system. The researcher can choose to completely randomize a particular amino acid position in the expressed protein, or flexibly tune the relative amounts of different amino acids at that position, as needs require (2). The combination of standard and custom Trimer Codon phosphoramidite mixes is a powerful research tool for anyone doing protein engineering research.