J. Beekwilder et al., A phagemid vector using the E-coli phage shock promoter facilitates phage display of toxic proteins, GENE, 228(1-2), 1999, pp. 23-31
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.