My protein of interest is an intrinsic disordeed protein. I tried BL21 (DE3) CBV-101, and C43. We have tried 18C overnight, 30C 5h and 37C 3h expression. The BL21 and CBV got killed because the protein leaked and is toxic to these two strains. C43 did not have a great amount of leak and is very healthy at the time of induction They all generate great IPTG inductions. Cells lysis was done by lysis buffer of T4lysozyme incubation with triton 100, protease inhibitor, PMSF 1mM, BME 1mM, 10%glycerol high salt buffer and room temperature shake for 15minutes, followed by sonication 40% amp 15s on 45s off for 5 cycles on ice. The cells were centrifuged at 18000rpm 1H. After His NTA column (by the way I am using the pSMT3 vector which has RBS followed by His-SUMO tag), 50mM,100mM, and 500mM imidazole elution were done. However, I do see my protein came out at 50mM using Western blot, and 100mM had more come out. Surprisingly no protein came out at 500mM imidaozle. While the100mM imidazole elution give me a relatively pure protein, the protein has three bands, a band of its own, a band right below(also same protein after western blot), and a band 10+Kda below. This lower band can also be my protein but because my antibody did not capture it, I wasn’t so sure but my antibody capture the C end so that could still be my protein. All three bands are tightly close and FPLC cannot be possible to purify them.
I am curious that at this situation, if I want to run a NMR do I have to have the pure protein without truncation? Or it is fine to probe the NMR? How do you avoid the truncation after all? Do you have experience on this type of protein and how do you handle them? do you think the truncation was caused by protease or RBS binding?
By the way, I do see these truncation bands even before protein extraction, which indicates the truncation comes before my protein extraction. The picture below is direct cook of Ecoli cells under laemmli buffer and BME. The left are uninduced and induced c43 at 37 and the right are 30C. The upper highlighted has two bands inside actually.
Hi,
If you can't avoid the cleavage completely (e.g. by adding more protease inhibitors) you could try to get those fragments separated, e.g. by size exclusion chromatography or ion-exchange which could also separate different forms of the same protein. Alternatively, you may use the fragments to identify (by MS Sequencing) stable fragments to express them separately and study their structures individually by NMR. This could give you also valuable information on its structure in comparison to the full length protein.
Hope that helps.
I think it would be interesting to figure out the amino acid sequence or range of your truncation construct by mass spectrometry analysis of the band excised from the gel subjected to trypsinization and liquid chromatography mass spectrometry-mass spectrometry or edman degradation sequencing.
For instance, Escherichia coli DNA Polymerase I is cleaved by the protease subtilisin into two fragments: the Klenow fragment and the 5'-3' exonuclease domain. The Klenow fragment contains 5'-3' polymerase activity, 3'-5' proof-reading exonuclease activity and it also has strand displacement capability as Klenow fragment can open molecular beacons. The only thing that the Klenow fragment cannot do that the full length protein cannot do 5'-3' exonuclease activity which is used by the full length protein to remove RNA primers in Okazaki fragments on the lagging strand in Okazaki fragments.
The 5'-3' exonuclease domain in the full length Escherichia coli DNA polymerase interferes with the 5'-3' polymerase activity and 3'-5' exonuclease activity making in vitro assays of the full length protein very difficult. It's like having your enzyme convert substrate into product and then degrade the product in an entirely other active site.
So a lot of biologist prefer to work with the Klenow fragment unless they are interested in 5'-3' exonuclease activity.
If you suspect that there might be some traction, you can perform mass spectroscopy to confirm it. From my perspective, it appears that there are numerous impurities present. To obtain your desired pure protein, you can rinse the Ni-NTA column with an excessive amount of wash buffer, preferably 20-50 mM Imidazole, depending on the concentration of the protein in the elution.
Thanks for all the answers. I finally found that these are not truncations but impurities that is strongly sticking with my protein. However, though part of the protein can be further purified through SEC column since the disordered protein ran out of the column in void volume. I can get around 200ug proteins from 1L ecoli culture, which is low in yield.
But most of my protein comes with another impurity protein together at fraction 8-12. I tried to further separate this fraction using imidazole in SEC with 500mM NaCl in HEPES buffer. The impurities do not separate from my protein. So The void volume does not have any of my protein and they still come in fraction8~12. Do you have any suggestions?
It seems like a lot of proteins in general are sticking to the Ni-NTA. Try cleaning, stripping, and regenerating the nickel NTA with NiCl2. Bind the supernatant to the resin and and do a 10-20 column volume wash with buffer with 1 M NaCl.
Nickel NTA binding is the most important step. But size exlclusion is the next step. How big in kDa is your target protein and what size is the gel filtration resin? We talking G-20, G-25, G50, G200?
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