Pentapeptide scanning mutagenesis: Random insertion of a variable five amino acid cassette in a target protein

Abstract

A new insertion method for probing protein functional organization was developed. The method relies on the random insertion of transposon Tn4430 and subsequent in vitro deletion of the bulk of the transposon after which a 15 bp insertion remains within the target gene. This results in pentapeptide insertions randomly distributed in the target protein. Characterization of 23 pentapeptide insertions in TEM-1 β-lactamase demonstrated the utility of the method. The phenotypes associated with the mutated β-lactamase proteins equated both with the sorts of local peptide structures in which the pentapeptide insertions occurred and their position in the three-dimensional structure of the enzyme.