Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage

Abstract

BackgroundFilamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles.ResultsThe amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags.ConclusionsOur results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies.

Multivalent pIX phage display selects for distinct and improved antibody properties

“…Here, the beneficial effects of pIX display was revealed only when pIX was utilized without the pelB signal sequence. Thus, the consequence of adding a pelB sequence could well be a re-routing from the YidC, the suggested route for native pIX13, to the SEC translocon. Moreover, the favorable properties appear to be unique to pIX, as M13 pVIII that also depends on YidC has been shown to be inferior to pIII in antibody affinity selection3637.…”

“…pIX and pIII are located at opposite tips on the virion, and the two proteins differ in their mechanisms of translocation to the E. coli inner membrane prior to phage assembly. Whereas pIII is synthesized as a precursor with a post-translationally processed signal sequence and targets the SEC translocation pathway12, pIX altogether lacks a signal sequence, does not undergo post-translational processing and appears to depend on YidC for periplasmic targeting1213. Early studies showed that an N -terminal GST fusion prevented pIX display, as pIX was found aggregated in the cytosol of the E. coli host14.…”

Next generation phage display by use of pVII and pIX as display scaffolds

“…In line with this notion, it has been shown that pIX spontaneously adopts an a-helical structure when inserted into artificial lipid vesicles [28]. However, a very recent report exploiting a YidC-deficient host for virion production provides compelling evidence that at least the pIX capsid is also dependent on the YidC transporter for inner membrane targeting and subsequent virion integration [29].…”

“…In one study, the 13 amino acid long AviTag was readily displayed on all copies of phage genome encoded pIX, (type 9 display, excluding wt complementation), but at the expense of markedly reduced virion production [103]. When a modified pIX harnessing affinity tags (T7-tag or HA) up to 36 amino acids supplied in trans from a plasmid and complementing an amber pIX M13 phage, this reduction in titer appeared lesser pronounced [29]. Notably, neither of these studies used an N-terminal signal sequence targeting of the pIX fusion, supporting our initial observation that efficient pIX display is not dependent on dedicated periplasmic targeting through a leader peptide.…”

“…The initial observation that pIX display required wt complementation to work [25,87], suggested that multivalent pIX display was unattainable. Two very recent reports challenge this view [29,103]. In one study, the 13 amino acid long AviTag was readily displayed on all copies of phage genome encoded pIX, (type 9 display, excluding wt complementation), but at the expense of markedly reduced virion production [103].…”