Surface Expression and Ligand-Based Selection of cDNAs Fused to Filamentous Phage Gene VI

Abstract

We describe a novel phage display system that affords the surface expression and hence affinity selection of cDNAs. The strategy is based on a new approach to functionally display proteins on filamentous phage through the attachment to the C-terminus of the minor coat protein VI. The utility of the method was evaluated using a cDNA library derived from the parasite Ancylostoma caninum. cDNA sequences were fused in each of the three reading frames to the 3'-end of the M13 gene VI expressed by a phagemid vector. Phages rescued from this cDNA expression library were subjected to biopanning against two serine proteases, trypsin and the human coagulation factor Xa. This led to the identification of cDNAs encoding novel members of two different families of serine protease inhibitors. The authenticity of the cDNA selected with trypsin as the target was demonstrated by purifying the encoded potent Kunitz-type inhibitor from an Ancylostoma caninum extract. The rapid isolation of specific cDNAs with the protein VI monovalent display system should facilitate the search for novel biologically important ligands.

Rapid Identification of Allergen-Encoding cDNA Clones by Phage Display and High-Density Arrays

“…Peptides coded by cDNAs generated by oligo dT-priming cannot be assembled in the phage coat by this approach since translation stop codons prevent the synthesis of N-terminal fusions to the coat proteins [13]. To overcome these problems, several approaches including direct fusion of inserts to the carboxyl terminus region of gene VI [14], gene III and gene XIII of filamentous phage [15,16], as well as the use of other phage systems [17,18] have been proposed and recently reviewed [16,19]. We devised an indirect strategy, fusing the Jun zipper to the gene for phage protein III to enable coexpressed N-terminally Fos-decorated cDNA products to be efficiently displayed on the phage capsid via Jun-Fos interaction [11,13].…”

Selecting Open Reading Frames From DNA

“…In general, attempts to display random ORFs on filamentous phage, such as those encoded by cDNA fragments, have not been very successful, notwithstanding the development of vectors in which random fragments are displayed at the C terminus of a Jun peptide that interacts with Fos displayed at the N terminus of p3 (Crameri et al 1994;Crameri and Blaser 1996), at the C terminus of p3 , p8 , p6 (Jespers et al 1995), or at the C terminus of an artificial protein that is able to replace p8 in filamentous phage (Weiss and Sidhu 2000), although successful display of a cDNA library was recently reported (Butteroni et al 2000). Greater success appears to have been achieved with -based vectors for cDNA display (Santini et al 1998;Beghetto et al 2001).…”

Profiling the autoantibody repertoire by serological antigen selection

“…In contrast to bacteriophage coat protein 3 (pIII) or 8 (pVIII), the phage coat protein pVI is not known to be involved in infection and has the characteristic that the C-terminus rather than the N-terminus is believed to be surface-exposed [28]. Therefore, the possible advantage of pVI for the display of cDNA repertoires is that the presence of stop codons does not prevent display.…”