Hello,
I'm relatively new to the field of Phage Display, and there are still points I don't perfectly understand. A bit of context : I'm using M13 phage along E.Coli for amplification/infection. The protein of interest (POI) is fused with pIII, and possesses a pelB leader sequence on the N-term. The classical phagemid / helper phage combination is used.
After an initial screening of a library, some clones were recently identified in my group. After sequencing, it appeared that the PelB leader sequence is absent from some of them.
If I understood correctly, the pelB leader sequence translocates the fusion-POI-pIII to the perisplam, where the virus particles will be assembled. If the pelB sequence is absent, the translocation process can not occur, preventing the assembly of the virus particle, and a novel infection.
Should I consider that these clones are false-positive ? Or can these particles be secreted ?
Best,
Cyril Legrand
It is possible that the fragment encodes a sequence that can act as a signal sequence for secretion. But I think it more likely they are weird false positives. However if upon retesting they behave correctly then they might be worth a followup.
If you are working with a system where you have both wt.gene III and the recombinant gene III in the system (see Fig.3.1 panel 8 in https://plueckthun.bioc.uzh.ch/wp-content/uploads/Publications/APpub0307.pdf for a schematic representation, you would end up with a phage that contains only wt. pIII, and therefore show good infectivity. You only get rid of these phages in the selection step.
Thank you for your answers.
Indeed it is straightforward to simply test these phages and check if they appear after another selection step.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning