Proteins are used in a number of industrial applications. Examples include their use as biocatalysts and in screening for novel drug candidates. Where suitable proteins do not exist in nature there is a tremendous need to engineer the production of novel proteins that can function in an industrial setting. This “engineering” of proteins is usually carried out by random mutagenesis. Randomised libraries are produced containing variations of a gene or gene fragment which are screened for novel activity. While a number of methods have been developed for introducing this diversity, none are without their drawbacks.

One widely used method is codon randomisation (saturation mutagenesis), which generates a library of mutants (a randomised gene library) in a single experiment. For full randomisation, key codons are typically replaced with NNN (64 sequences) or NN^G_CorT (32 sequences). This obligates cloning of redundant codons alongside those required to encode the 20 amino acids. As the number of randomised codons increases, there is therefore a progressive loss of randomisation efficiency; the number of genes required per protein rises exponentially. The redundant codons cause amino acids to be represented unevenly, and the organisation of the genetic code makes it impossible to encode functional subsets of amino acids (e.g. polar residues only) in a single experiment. Scientists at Aston have previously developed a method of randomisation which eliminates this redundancy and bias. However, even using this method the production of more than two contiguous randomised codons proved problematic.

Further work at Aston University has now led to the development of an improved protein randomisation method which virtually eliminates the presence of unwanted sequences whilst also enabling the production of contiguous randomised codons with little additional effort. The method uses simple and easily performed cleavage, ligation and amplification reactions and can be carried out sequentially to produce contiguous randomised codons if required. Use of the Aston method should substantially improve the efficiency and reduce the cost of producing randomised proteins for screening for novel functionality.