Antibody engineering using very long template-assembled oligonucleotides

Abstract

We have previously constructed single-stranded DNA fragments up to 361 nucleotides long by the ligation of shorter synthetic fragments assembled on a closely homologous template and used them for site-directed mutagenesis. Here we have extended the utility of such long oligonucleotides for protein engineering in two ways. First oligonucleotides have been assembled on a template to which they show only relatively weak sequence homology (<70% identity), allowing conversion of genes encoding rather distantly related proteins. We have converted a gene encoding human light chain constant domain from the kappa to the lambda isotype, which share <40% amino acid identity. Second, libraries of random mutants were constructed within antibody variable domains with up to 20 targeted codons separated by up to 186 nucleotides. These libraries were created by template-directed assembly of degenerate oligonucleotides followed by PCR amplification and subcloning. Nucleotide sequence analysis of clones demonstrated that this is an effective method for the simultaneous random mutagenesis of several codons distant within the target gene. This should provide a useful tool for modifying the ligand binding properties of proteins, e.g., the antigen binding affinity and specificity of antibodies.