Development and applications of codon scanning mutagenesis: A novel mutagenesis method that facilitates in-frame codon mutations

Abstract

The ability to create protein variants is a very valuable tool in biochemistry. Information about mechanistic roles of amino acid side chains, protein topology and binding can all be obtained. Methodologies to mutate proteins also allow for new catalytic activity to be achieved. While the routinely used methods to alter a protein sequence have proven to be useful, to some degree each of these methods requires some knowledge of protein structure to determine the site of mutation. Further, the routinely used methods also only allow for a specified site to be changed to a pre-determined residue (directed by oligonucleotides) or for multiple random sites to be changed to a non-specified residue. This dissertation focuses on the development of a method that allows for a new defined amino acid to replace a native amino acid at a random location within in the protein. To introduce mutations at random locations within a protein coding sequence, three steps need to be accomplished. First, the coding sequence needs to be randomly digested on both strands; second, three nucleotides (a codon) at the digestion site need to be removed; and last, a new specified codon inserted. This process results in the replacement of a random codon with the new defined codon. To direct a mutation at a random location, the unique properties of a transposase/transposon are used to create both the double strand break and removal of three nucleotides. The insertion of the new defined codon is introduced using a linker sequence that when inserted in the correct reading frame a selectable phenotype is produced. This process has been termed Codon Scanning Mutagenesis (CSM). The advantages of this method over current mutagenesis methods are (1) knowledge of structural information is not required, (2) oligonucleotides are not required to introduce the mutation and (3) the mutagenesis method allows for every amino acid to be mutated regardless of the DNA sequence. Further, this method allows for any natural and unnatural amino acid to be inserted at the mutation site, as well as the ability to create mutational mixtures or introduce multiple user defined mutations.