A couple of people asked why the GST/His steps. The only way I can expressed this protein is by co-expression. Co-protein is GST tagged and my protein of interest is N-term His and C-term StrepTag. Even with the coexpression, there is a major degradation product, the reason why for the strep tag, but now it is not being removed by the column. I have tried size exclusion (140kDa vs 100kDa does not work) and MonoQ (pI are near identical). I have been using HABA for regeneration. I will try some of the suggestions to try and rescue it. Summaries are presented below.
A summary of answers: 1) We use a Streptactin resin from IBA and they recommend regenerating with HABA, which is non-destructive and I believe can be used many times (we've done greater than 7 times I believe?). They say their linker is more affected than GE's version so NaOH is quite destructive and cannot be run more than ~ 3 times with their resin. Perhaps GE resin is also affected more by NaOH? HABA will displace any biotin. 2) I use 5 ml StrepTrap columns from GE and these columns work almost indefinitely with reproducible results if the following trick is used .I pre-clear biotin from my lysate with a pinch of avidin (Sigma #A9275-25MG) and find that this works really well. You could try adding a small amount of avidin to your protein sample after your Nickel column. After each run, I wash the column with 3 CV 0.5 N NaOH, 10 CV water, 10 CV 1 mM HABA (Sigma H5126-25G) containing buffer (pH 8) and finally equilibrate with 10 CV binding buffer. In the past, I used to see a gradient of pale orange at the top of the column and dark orange at the bottom as the column got older, presumably because of sites inactivated by biotin. Surprisingly, this phenomenon went away after avidin treatment suggesting that it might be possible to rescue old columns inactivated by biotin although I haven't rigorously tested this. 3) We have been using Strep-Tactin SuperFlow (high capacity) columns more than 100 times, even when run under preparative conditions with protein extracts from E. coli fermentations. Biotin binding to the engineered streptavidin is not irreversible but can be cured by extended washing. To check the column status I recommend our HABA binding procedure described in: Schmidt, T. G. M. & Skerra, A. (2007) The Strep-tag system for one-step purification and high affinity detection or capturing of proteins. Nat. Protoc. 2, 1528-1535. 4) i am not the last authority on this, and really, IBA have in the past been very helpful (http://www.iba-go.com) with information. But here my 2 cents on this: i would give it a 20 times possibly. you CAN even apply lysate directly and iba describes on their pages how you avoid biotin contamination with addition of streptavidin to your sample. streptavidin catches all the free biotin, but does not bind the strep tag. done it and works real well, too ... 5) I used the Strep-Tactin resin from IBA Bio TAGnologies, 2-1201-010, and would regenerate it after each use with 2 mM HABA (Sigma H5126-25G). I did not observe a decrease in affinity over multiple uses. Also, I only used the one column and obtained 99% pure protein.
