Hello,
I've got several VHH sequences after phage pannings and cloned them into pET28a vector to express. After transforming them into BL21(DE3) E.coli strain and inducing with IPTG, I found almost all of them hardly expressed, which shared a same origin germline IgHV.
Could someone explain that whether the sequence of these VHHs disrupt the expression? Or if there are some types of VHHs hard to express in E.coli system naturally? Should I change my expression system, for example, in HEK293 system?
Thanks in advance for all answers.
Lyron
Yes, the individual amino acid sequence can very strongly affect levels of functional expression. However, this will usually lead to a large proportion of the protein ending up in the insoluble fraction, not in the protein not being expressed at all. However, poorly folding antibody fragments can be toxic to the bacteria, resulting in poor growth until the culture gets overgrown by bacteria that managed to get rid of the offending gene. If the problem lies within the protein sequence, going to a mammalian system might somewhat overcome the production problem, but still leaving you with an unstable and/or aggregation prone protein.
To properly diagnose your problem we need to know more about what you actually are doing.
Can you share the germline sequence that is causing the problems? Have you seen other nanobodies that express well in exactly the same system?
How are you expressing your VHH - cytoplasmic or periplasmic expression? Are you looking at soluble expression only or also checking for insoluble protein? Can you be sure that in your vector prep, you do not have picked up a mutation in the 5' untranslated region, resulting in poor transcription or translation?
Certainly need more information here. I normally transfer nanobodies over to pET22b as there is a direct NcoI/NotI cloning compatibility that retains the pelB periplasmic secretion signal sequence. The benefit of this system is that we have a streamlined method to screen clones in 96-well blocks and use the osmotic shock supernatant for functional screen to verify fidelity to target. It is typical to see yields of 5-50mg/liter with nanobody expression. You may have the atypical clone on occasion that does not express well, but I have rarely had this issue. My guess is either your cloning did not work well or your purification method isn't working. Happy to help diagnose with additional information.
Jy Zhao, Here you go:
Here is the general protocol, which is adapted from the Pardon et al. (2014, Nature Protocols) paper.
1. In a 96-well deep block (2ml wells), add 1ml of Terrific broth per well.
2. Using sterile pipet tips, pick clearly isolated colonies into each well of the plate.
3. Grow the cultures for 5-6 hours until dense (I don't measure OD) at 37C, 300rpm shaking.
4. Remove 100ul of the culture into a sterile 96-well plate; this is the archive plate. Store at 4C for up to 5 days.
5. Prepare a 10mM IPTG solution in the same media.
6. Add 100ul 10mM IPTG solution per well and cover plate with breathable sealer and continue growing at 37C, 300rpm shaking overnight.
7. After overnight growth, spin the block down at 1500xG at 4C for 20min.
8. Gently discard the supernatants, using care to not dislodge the pellets.
9. Place the block in the -80C for 1hr.
10. After 1hr, bring the block to the bench and allow the block to warm for 30mins.
11. Add 500uL 1xPBS to each well and shake at 1500rpm for 1hr. Ensure that all of the pellets are homogenously resuspended. This shock step is sufficient to release enough nanobody for ELISA screening.
12. Spin the block for 20min at 2500xG, 4C.
13. The supernatant is ready for transfer to the antigen plate, 100uL per well. **during this entire process, I will have my pre-coated antigen plate ready to go, blocked with either 2% non-fat dry milk or 2% bovine serum albumin.**
14. Incubate the supes for 1-2hrs at room temp or overnight at 4C.
15. Wash the plate, then add 100uL your choice of detection antibody. I use the anti-alpaca VHH specific (HRP conjugated) from Jackson ImmunoResearch at 1:1000 in 1% BSA/PBS for 1hr at room temp with shaking at 600rpm. (https://www.jacksonimmuno.com/catalog/products/128-035-232)
16. Wash plate and develop using TMB or other chromagen of choice. Hope this helps. Let me know if you have any questions. You can reference the Pardon paper or my recent Covid paper (https://www.nature.com/articles/s41598-020-79036-0) Best, Thomas
Jy Zhao yes, for this small scale it is a freeze-thaw. I have done the osmotic shock method and I don't see a difference for screening ELISAs. The amount is on the microgram scale so I don't know how useful it would be to try and purify.
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