I'm performing an antibody phage display with a VHH library and I consistently get frameshift mutants (mainly frame +2) after biopanning. I'm using TG-1 cells for amplification of phagemids and VCSM13 as helper phage. Biopannings are performed in target protein-coated immunotubes and PBS-milk is used as blocking agent. I have tried to coat the immunotubes with different protein concentrations (10-100 ug/mL in carbonate coating buffer) with the same results. Also tried the microtiter plate format. When I analyze the original library, all the clones are in the correct frame. I would appreciate any explanation or suggestion. Thanks!
Hi, that is nothing about the selection itself but the tendency of propagating transcription errors in expression systems with high selective pressure.
Having no idea about how you rescue phage, but my usual advice is:
- 20-30 deg C after re-infection
- rich media in propagating after selection
- best seems to be growing infected bacteria on dYT (+glucose? in particular for IPTG induced systems) agar plates at 30 deg
- induction/expression of antibody only when absolutely necessary (some systems still produce enough (-> phage prep on protein gel (or even better western blot), compare P3-antibody fusion to P3)
Regards
Michael
Hi Daniel,
that is indeed curious, I can only think of the two following factors contributing to this effect:
1. The Helper Phage Infection Efficiency:
It could be that the VCSM13 helper phage might be contributing to the observed high mutation rates. To address this, I suggest ensuring you are using a fresh, high-titer VCSM13 stock. Sometimes, issues arise from aged or low-quality helper phage stocks. As an additional step, it might be worth considering a switch to a different helper phage, such as M13KO7, just to ascertain if the helper phage itself is the culprit.
2. The Bacterial Host Strain:
TG-1 cells are commonly utilized in phage display, however, they may not be the optimal strain for your specific application. It is worth it to verify that the strain you are using is both competent and healthy, as stressed or suboptimal bacterial cultures can introduce mutations. Experimenting with another E. coli strain, like XL1-Blue or SS320, could potentially provide valuable insights.
I find myself leaning towards the first point, suspecting that the helper phage you are currently using might be more likely to introduce some form of frame shift mutations.
Cheers
Stöpa
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