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

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 Not I 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.