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fd and IKe are two similar filamentous phage which infect their hosts by means of pili found on the host membrane: fd infects bacteria bearing F pili, whereas IKe infects bacteria bearing N or I pili. Infection is mediated by the gene 3 protein (g3p), which of the nine proteins found in both phage is the most diverse. Previous attempts to incorporate g3p from one phage into the other by complementation...
We have constructed phagemid and phage-based vectors which can be used for both direct (T/A) and ligation-independent cloning (LIC) of PCR products for surface display of encoded peptides/proteins fused with the gIII protein of the filamentous bacteriophages M13 and fd-tet. The vectors harbour a DNA cassette consisting of the lacZα fragment inserted between the + 2 and + 3 codons of gIIIp. The lacZα...
A method was developed to clone linear DNAs by overexpressing T4 phage DNA ligase in vivo, based upon recombination deficient E. coli derivatives that carry a plasmid containing an inducible T4 DNA ligase gene. Integration of this ligase-plasmid into the chromosome of such E. coli allows standard plasmid isolation following linear DNA transformation of the strains containing high levels of T4 DNA...
We describe the use of transcriptional fusions to the phage shock protein (psp) promoter. These fusions are expressed only when cells are infected by filamentous phage. In an application, the psp promoter was fused to the protein coding part of filamentous phage gene III (gIII). Protein III (pIII) is needed to complement mutant f1 phage containing a deletion of gIII, but its synthesis also renders...
A mutation strategy which utilises phage display technology and the Escherichia coli mutator strains, mutD5-FIT and XL1-RED, was applied to a Hepatitis B (HepB) specific single-chain Fv (scFv) to incorporate random mutations throughout the gene. Messenger RNA from a hybridoma producing antibodies against HepB was isolated, reverse transcribed and used as template for the production of scFv. Following...
A synthetic gene for hirudin was ligated into phagemid pCANTAB5E. This construct allows production of either soluble hirudin or phage having hirudin displayed on the surface. Similarly, hirudin variants with extensions either at their N- or C-terminus were generated. The genes were expressed in their soluble form in a non-suppressor strain of E. coli. Periplasmatic fractions were evaluated in standard...
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