A novel random mutagenesis method and the incorporation of an isotopic label into a protein

Abstract

There are limitations on the current methods used to diversify protein sequences, such as the inability to scan mutations throughout an entire protein. We have developed a novel method, “Codon Scanning Mutagenesis”, which allows for rapid and complete scanning of a protein sequence with the use of elementary molecular biology techniques. This method is made possible by a modified transposon mutagenesis approach that randomly targets DNA sequences. This transposon allows for random insertion of a NotI restriction site, which is unique to the DNA sequence to be mutated. The randomized insertion of this restriction site thus allows for the insertion of a linker with strategically placed type II restriction sites. The properties of the type II restriction sites allow for a codon to be replaced with a new codon. We are specifically interested in the random insertion of TAG stop codons which code for unnatural amino acids carrying photoaffinity labels that can be used to probe protein-protein interactions. Finally, we show that the detection of photo-crosslinked protein-protein interactions can easily be done by mass spectrometry studies when the deuterated p-benzoylphenylalanine is incorporated as an isotopic label. This method will allow for obtaining structural information on multi-component protein complexes, provide insight into the active site residues, or even trap receptor ligands at the cell surface.