Unexpected frameshifts from gene to expressed protein in a phage-displayed peptide library

A library of long peptides displayed on the pIII protein of filamentous phage was used in biopanning experiments against several protein targets. We find that a large percentage of phage clones that bind specifically to a target contain peptide-encoding genes that do not have an ORF. Instead, the reading frame is either interrupted by one or more nonsuppressed stop codons, or a post-transcriptional frameshift is needed to account for the expression of the minor phage coat protein pIII. The percentage of frameshifted clones varies depending on the target. It can be as high as 90% for clones specific for soluble forms of certain cytokine receptors. Conversely, biopanning against four mAbs did not yield any frameshifted clones. Our studies focused on one clone that binds specifically to rat growth hormone binding protein (GHBP) yet does not have an ORF. A secondary peptide library containing random mutations of this sequence was constructed and panned against GHBP to optimize and correct the reading frame. In the last round (round two) of panning with this library, none of the phage clones that bound to GHBP had an ORF. However, careful analysis of these clones allowed us to design a synthetic peptide capable of binding to GHBP. The results of this study indicate that ORFs are not required to obtain gene expression of the minor coat protein of filamentous phage and suggest that some ORF(−) clones may have a selective advantage over the clones having ORFs.