A phagemid vector using the E-coli phage shock promoter facilitates phage display of toxic proteins

Phage display is a powerful tool with which to adapt the specificity of pro tease inhibitors. To this end, a library of variants of the potato protease inhibitor PI2 was introduced in a canonical phagemid vector. Although PI2 is a natural trypsin inhibitor, we were unable to select trypsin-binding va riants from the library. Instead, only mutants carrying deletions or amber stop codons were found. Bacteria carrying these mutations had a much faster growth rate than those carrying the wt PI2-encoding gene, even when the pr omoter was repressed. To overcome these problems, two new phagemid vectors for g3-mediated phage display were constructed. The first vector has a lowe r plasmid copy number, as compared to the canonical vector. Bacteria harbor ing this new vector are much less affected by the presence of the PI2-g3 fu sion gene, which appears from a markedly reduced growth retardation. A seco nd vector was equipped with the promoter of the Escherichia coil psp operon , instead of the lac promoter, to control the PI2-g3 gene fusion expression . The psp promoter is induced upon helper phage infection. A phagemid vecto r with this promoter controlling a PI2-g3 gene fusion did not affect the vi ability of the host. Furthermore, both new vectors were shown to produce ph age particles that display the inhibitor protein and were therefore conside red suitable for phage display. The inhibitor library was introduced in bot h new vectors. Trypsin-binding phages with inhibitory sequences were select ed, instead of sequences with stop codons or deletions. This demonstrates t he usefulness of these new vectors for phage display of proteins that affec t the viability of E. coil. (C) 1999 Elsevier Science B.V. All rights reser ved.