Identification and characterization of mutant clones with enhanced propagation rates from phage-displayed peptide libraries

Abstract

A target-unrelated peptide (TUP) can arise in phage display selection experiments as a result of a propagation advantage exhibited by the phage clone displaying the peptide. We previously characterized HAIYPRH, from the M13-based Ph.D.-7 phage display library, as a propagation-related TUP resulting from a G→A mutation in the Shine-Dalgarno sequence of gene II. This mutant was shown to propagate in Escherichia coli at a dramatically faster rate than phage bearing the wild-type Shine-Dalgarno sequence. We now report 27 additional fast-propagating clones displaying 24 different peptides and carrying 14 unique mutations. Most of these mutations are found either in or upstream of the gene II Shine-Dalgarno sequence, but still within the mRNA transcript of gene II. All 27 clones propagate at significantly higher rates than normal library phage, most within experimental error of wild-type M13 propagation, suggesting that mutations arise to compensate for the reduced virulence caused by the insertion of a lacZα cassette proximal to the replication origin of the phage used to construct the library. We also describe an efficient and convenient assay to diagnose propagation-related TUPS among peptide sequences selected by phage display.

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage

“…We returned to an approach we previously reported [41] to identify additional fast-propagating clones from the Ph.D.-7 library. First, three rounds of serial amplification of the phage-displayed peptide library without any panning steps ( i .…”

“…Fast-propagating clones from the Ph.D.-7 library were identified as previously described [41]. Briefly, the Ph.D.-7 library was serially amplified three times.…”

“…To isolate the T5091C mutation from the double mutant Ph-HAFPHLH, which contains both the T5091C mutation and a G6793Δ deletion [41], the Acc65I-to-EcoRI fragment of Ph-HAFPHLH (containing the T5091C mutation and the displayed peptide HAFPHLH) was ligated to the EcoRI-to-Acc65I fragment of M13KE (containing a G at position 6793). The Acc65I-to-EcoRI fragment was obtained by PCR using the single-stranded Ph-HAFPHLH genome (purified from a single plaque) as the template and the primers and which annealed upstream of the EcoRI site (on the coding strand) and downstream of the Acc65I site (on the noncoding strand), respectively.…”

“…coli cells significantly faster than M13KE phage as well as the pool of library phage from which they come [40,41]. The vast majority of these phage clones have single mutations or deletions in the 5’-untranslated region (5’-UTR) of gene II.…”

SABinder: A Web Service for Predicting Streptavidin-Binding Peptides

“…TUP can be divided into two types, propagation related and selection related. A propagation-related TUP can arise in the output of phage display because it has a propagation advantage [7, 8]. In contrary, a selection-related TUP can sneak into the results of biopanning as a result of reacting with other components of the biopanning systems instead of the target [6, 9].…”