In summary, during the expression process, my protein underwent significant translational interruption, leading to the production of a large amount of truncated target protein. I heterologously expressed the human cGAS protein in Rosetta DE3 cells. The expression was induced at 220 rpm, 18°C, with a final IPTG concentration of 200 µM for 20 hours. However, after ultrasonic lysis, SDS-PAGE electrophoresis revealed the abundant expression of an approximately 33 kDa N-terminal cGAS protein.
The reason I believe that the truncated cGAS protein was expressed is that this 33 kDa protein could be adsorbed onto and eluted from a nickel affinity chromatography column, cleaved by ULP1 enzyme, and my target protein’s N-terminus happens to contain a 6×His tag and a Sumo tag.
So the question now is, why is my expression host producing a large amount of truncated target protein? Are there any potential solutions? Thank you all!
Hi Yiheng,
What MW did you expect for this protein? Is there a disordered region within it that might be cut by E. Coli proteases to leave a 33 kDa domain? Did you add Ulp1 enzyme to it? Ulp1 is a yeast protease so it should not be present in Rosetta cells unless you put it there. Even though it is not present another protease might cut in the linker region between SUMO and your protein.
You can run a gel including the pelleted cell debris to see if there is an insoluble full length fragment in the pellet. If there is you can try adding detergents to solubilize it. You can try adding PMSF or protease inhibitor cocktail to stop proteases from cutting your protein into smaller pieces during purification. If all else fails you can make another construct using different fusion tags such as a C-terminal His tag - that would enrich your sample for proteins that include the whole sequence.
Yiheng, as it so happens I just published a paper on this issue. See the following publication: https://pubmed.ncbi.nlm.nih.gov/39692120/. If I am understanding your problem correctly, you are seeing the expression of a truncated protein corresponding to the size of your protein of interest that has His6-SUMO attached at the N-terminus.
I found that Shine-Dalgarno-like sequences (i.e. GGNGGN) within the SUMO tag promote internal translation events. If this is the issue, you can either 1) use SUMO^NIT that mutated those Shine-Dalgarno-like sequences or 2) mutate the methionine(s) at the N-terminus of your target protein. Gly-Gly motifs tend to give this issue in general if there is a downstream methionine, depending on mRNA secondary structure and whatnot. Good luck!
Yiheng, the plasmid encoding the mutated SUMO is now available on Addgene: https://www.addgene.org/Venkat_Gopalan/. Good luck!
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