Hi guys,
I'm currently struggling with protein expression... my POI shows very low yield and it seems like the protein expression induction is not very efficient... I already sequenced my plasmid, tried different IPTG concentrations (300nM - 1mM), induction times (between 1 and 3h at 37, 30, 20 degrees Celsius), ODs (0.5-1.0), etc... When I increase my culture to 2L I get a correct band in SDS-PAGE and a small peak (300 mAU) during protein purification using the AKTA (whilst getting nearly 2000 mAU for my other POIs). Unfortunately, the protein amounts are not sufficient.
Did someone have similar problems and can suggest another way to solve my problem than still increasing my culture volume?
Thanks for your help.
It really sounds like you need to completely start over. Start with fresh reagents and fresh cells. Make sure the bacterial cell you use for expression is the right strain and clonally pure. Make sure the plasmid you are using has the correct promoter sequence, and also clonally pure. Make sure the culture media you are using is correct. Make sure the inserted gene (of the protein you are purifying) is in the correct reading frame. Make sure you are using the right antibiotic in selecting for your clone. Etc., etc. Bonne chance et au revoir.
Are you sure the protein you are expressing is not toxic to the host? We have had this impression a long time ago and resorted to growing the bugs as a lawn on plates, where, for some reason it wasnt so much of a problem.
Thank you for your answers!
Well, I can see that they're still growing after IPTG induced protein expression... So I don't think it's toxic. I'm really confused that the other POIs which are in the same plasmid, respectively, look perfectly fine. I always use fresh transformants and fresh media... already tried BL21 and BL21 pLysS as hosts but I still don't get what I'm aiming for.
Inclusion bodies?
Tried dissolving part of the pellet after the extraction procedure in SDS loading buffer and running that alongside the extract?
There seem to be no inclusion bodies according to my SDS-PAGE analysis...
Curiouser and curiouser!
Have you assessed expression of the POI in the bacterial supernatant and in a whole cell lysate (boiling up some bugs in SDS page load) prior to extraction to establish if its an expression or and extraction problem?
Have you shown that your plasmid contains a single insert in the correct orientation (we had problems with a tail to tail double insert a long time ago and that affected expression).
How are you preparing the bug extracts?
Yes that's what I did in the first place... Which is why I think it's an expression problem. According to my PI the plasmid looks perfectly fine.
I re suspend the pellet in 1x PBS containing EDTA-free protease inhibitor and then I sonicate for 5 min at 4 °C with an 20% amplitude (works out for all the others...). Thus, I centrifuge at 20,000 rpm and keep the supernatant which should contain my POI. But I only get weak signals and none in the insoluble fraction... It's quite odd but maybe I'll try another strain or scale further up. Seems like I'm wasting my time...
P.S. As in:
https://gtmb.org/volumes/Vol10/01.%20Chen%20and%20Texada,%201-12.pdf
Hi, Lena try the Rosetta II cells if your gene of interest is a human gene or from higher eukaryots. It contains plasmid with rare tRNAs. This can help.
Try to use autoinduction media; TB or 2xYT. Lower temperature (18-16°C) during expression. What tag do you use? Expression plasmid?
Good luck!
Thank you, Geoff!
Yes I was thinking about using Rosetta, too. I use a His-tag and pET28a(+) plasmid... So actually it's not a human gene but I'll give Rosetta a shot... Thanks!
this link is useful
https://rockland-inc.com/protein-expression-tips.aspx
regards
Follow this link
https://www.google.com/amp/s/www.researchgate.net/post/How_can_I_increase_the_protein_yield/amp
regards
You can use different incubate temperature and different concentration of IPTG for introduce more your target protein but the problem is it can really take time. Also you can increase time of inducing
Well, U have the same problems which I have faced in my constructs. I suggest that try to minimize lost during your downstream process and protein purification. In protein purification, you can be use eppendorf filled with His beads. First stage is concentrate your crude protein in less volume starting from first stage of purification onwards. I even optimized with harvested from 200 ml LB. and final 40 ul purified sample had observed protein band in SDS-PAGE. All the best and welcome for queries.
Milad, yes I did sonicate.
Thanks for your help, I'll consider it for my next tries!
Make sure to use protease inhibitor during cell lysis or sonication. 0.8-1mM PMSF should be good.
Did you already try Studier's autoinduction medium? You can have 10x biomass for the same culture volume, and almost always you will have better inductions than with IPTG (or at least, the same level of induction). Forget to follow the OD of your culture while waiting for adding IPTG, you can sleep at home while induction occurs "spontaneously".
The only problem with Studier's media is to find at your lab every reagent needed for the "trace elements" solution. You can do a preliminar test with just the corresponding concentration of Fe, and do not forget Mg!
- Badges
- Science method