Hi all, I am currently working with a bacterial membrane protein with intentions on solubilizing it through styrene-maleic acid (SMA) copolymers. Previously, I'm able to express this protein to good yield in the pET-21a(+) vector with a C-terminal 6xHis-tag, however, I am not able to purify this protein using IMAC due to unfavorable interactions between the copolymer and nickel resin. Because of this, I wanted to use a metal-free affinity purification technique, such as the twin-strep system. I constructed a new vector in the same pET-21a(+) vector, but with an added N-terminal twin-strep tag, but this construct did not express at all with different media, temperatures, times, etc. I constructed another vector, but this time adding a twin-strep-SUMO N-terminal tag, hoping that the possible disorder from the twin-strep tag would be alleviated with the added SUMO tag, however, I am getting the same issue with the protein not overexpressing. Does anyone have any solutions to this? I am currently growing in 1L TB media, BL21 cell line, 37C growth with 18C overnight induction with IPTG. It is possible that adding the N-term tags disable the protein from being expressed, since without it, the protein is expressed to good yields.
Adding N-terminal tags to your membrane protein construct is causing expression issues. This is not uncommon, as the addition of tags can affect protein folding, stability, and expression levels, especially for membrane proteins. Here are some steps you can take to optimize your expression:
- Promoter Strength: Adjust the strength of the promoter. You can use a weaker or tunable promoter (I have successfully expressed a toxic viral protease in the Lemo-21 strain by optimizing the expression with L-Rhammanose) system to regulate expression levels. This may help prevent overexpression issues.
- Chaperones: Co-express chaperones or folding factors that aid in membrane protein folding and stability. Chaperones like GroEL-GroES or DnaK-DnaJ-GrpE are commonly used.
- Cell Strain: Consider using different E. coli strains, such as C41(DE3) or C43(DE3), known for improving membrane protein expression. You might want to read this if you haven't already (Wagner, S., Klepsch, M. M., Schlegel, S., Appel, A., Draheim, R., Tarry, M., Högbom, M., Slotboom, D. J., & Persson, J. O. (2008). Tuning Escherichia coli for membrane protein overexpression. Proceedings of the National Academy of Sciences of the United States of America, 105(38), 14371-14376. https://doi.org/10.1073/pnas.0804090105)
- Solubilization and Purification: If you're still encountering difficulties, you might explore alternative solubilization and purification methods specific to membrane proteins. Detergent-based approaches, like the use of mild detergents such as DDM or digitonin, can sometimes be more effective.
Do let me know if it was helpful or not. All the best.
Like Aditya had said, it looks like adding N-terminal tags to your membrane protein construct caused the expression issues. Also, according to Aditya's suggestion, it may also be wise that you explore alternative solubilization and purification methods. DDM works well for many different membrane proteins in my hands.
If you could not make it work after trying all the suggestions mentioned, you may consider trying this protein production core facility (https://tamu.corefacilities.org/service_center/show_external/5805) which has served many labs for their difficult-to-express protein projects. Best wishes.
- Badges
- Science method
- Similar topics
- Biological Science
- Ecology