Simple Procedure for Creation of In-Frame Deletion Mutations Throughout an Open Reading Frame

A general method is presented for randomly mutagenizing open reading frames (ORF) to generate in-frame deletions and insertions. The protocol requires expression of the ORF of interest as a hybrid ORF-beta-galactosidase fusion protein. This allows colorimetric screening for beta-galactosidase activity during subsequent mutagenesis steps. Consequently, proteins with no suitable phenotypic selection or screening properties can be readily screened for mutations that disrupt and subsequently restore the reading frame of the hybrid protein. In addition, this system provides gene expression for subsequent biochemical analysis of the mutant proteins. The bovine papillomavirus type 1 (BPV-1) EI ORF has been mutagenized using this method as an example.