large protein degradation - during baculovirus purification (Jan/16/2007 )
I'm currently having difficulties purifying a very large protein (>200kDa) using Baculovirus. I have Flag and 10xHIS tags, but first problem is a lack of binding to a HIS column, and secondly the majority of products I'm purifying are significantly smaller than 200kDa, but cross react with 2 specific antibodies and the Flag antibody suggesting that they are degradation products. They are present as these sized fragments in the original input so the degradation is occuring inside the Sf9 cells - I am using Roche complete protease inhibs (EDTA free) + PMSF + chymstatin + aprotinin as inhibitors.
The Flag peptide is at the C-terminus so full length protein is being generated, but it doesn't remain intact. I think the lack of binding to the HIS column is because this end of the protein is cut off.
i assume His tag should be at Nterminus cause you said you suspect degradation due to not his binding.
Well did you do a time course production?
Usually 72h after infection cellls are collected. But with my SF9 cells, 24h give of course lower production, but degraded forms are reduced significantely.
Second question : what is the passage number of your cells?
Do you scrape them or detach by pipeting?
The HIS tag is at the N-terminus. The Flag tag is at the C-terminus and I can get good purification with this, but I'm worried that its not full length because it still won't bind to the HIS column, and doesn't X-react with the anti-HIS antibody. The degradation products present in the HIS purification are not present in the Flag purification but I think this is because they don't bind the FLAG resin, so I don't get very much protein out of the Flag purification.
The timecourse of protein collection suggestion sounds like a good one, so I will give it a go.
I'm not sure of the passage number of the Sf9 cells, they are a lab stock but they are looked after properly and used to purify other proteins with no problem. The cells are grown in suspension so are collected by centrifugation and the pellets frozen (I have tried using fresh pellets too with same result).