Background: I am developing an RdRp assay for Dengue virus-3 and need to express and isolate the NS5 protein's RdRp domain (~101 kDa). My expression vector includes a GST tag followed by a 6x His tag and then the dengue NS5 sequence. Sequencing has confirmed the clone.
Expression Conditions: The NS5 gene was cloned into the pET-41a vector, and expression was carried out in BL21(DE3) cells. Induction with IPTG (0.5 mM) at 32°C was performed for 11 hours, with samples taken every 2 hours.
Observation: During SDS-PAGE analysis and Western blotting with His antibody, I observed a prominent band around 35 kDa that increased with the induction time. This band is also present after purification using Nickel NTA resin. The expected combined size of GST and 6x His tags is around 26 kDa, while the NS5 protein size is 75 kDa.
What could be the identity of the 35 kDa band observed during induction and purification? Could this band represent a truncated protein? How common is extensive protease cleavage in such expression systems?
Any insights or suggestions to identify and troubleshoot this issue would be greatly appreciated.
I see 2 bands. Proteolysis is a plausible explanation. Since your antibody recognizes the His tag, which is near the N-terminus of the construct (GST is ~ 26 kDa), these 2 bands would probably include some of the GST sequence. You could test the idea by doing a Western blot with an anti-GST antibody. You might also try running SDS-PAGE and staining the gel to see whether these bands are abundant enough to cut out for mass spectrometry to identify them. With sufficient information about the cleavage sites from MS, you might be able to redesign the construct to eliminate protease hypersensitive sites.
Also consider whether there is anything you can do to reduce the proteolysis when preparing the cell extract, such as including a protease inhibitor cocktail, keeping the temperature low, working quickly, or changing the expression strain.
You really stayed there to plot 11h of 37°C overexpression?
In BL21 I did not observe any notable degradation thus far. Even then, 100% cleavage is not to be expected, I don't even get that with TEV protease cutting its fusion partner in vivo, prior lysis, as a detectable amount of freshly expressed uncut protein is always present. If there is a full length protein, you should be able to detect it in the Western Blot.
Sometimes proteins run at a slightly different molecular weight in SDS-PAGE than they should, due to folding or composition, but not to this extend. When doing lysis, always add an appropriate amount of protease inhibitor cocktail as Adam mentioned. Still, this doesn't seem to be the issue here, degradation tends to produce distinct bands as well as smears.
Something with your construct is off. Obviously, it doesn't produce the desired protein. I suggest reassessing the construct, testing the cloning steps in-silico and then doing it all over again. Especially complex cloning reactions tend to be more magic than science (yours doesn't seem that complex) and at times DNA tells you yes and the protein tells you no, for some inexplicable reason. Sadly, during cloning you work blindly as you can't see any of the stuff you're working with. Especially an impure plasmid backbone can make you question your sanity and make you lose weeks of your life.
35 kDa band (looking at the fig, its below 35 kDa molecular marker band, around 26-27 kDa) you observed during induction, possibly is GST-His. Its common to get GST protein in GST-protein of interest purification. Did you purified the protein using affinity chromatography before doing western? How you perform the lysis??