Labwork has been frustrating lately. My favorite protein (MFP) has been less than cooperative over the years. First, it was a royal pain in the butt to purify to any reasonable degree. At best, I had to settle for a co-purified complex that was for the most part functional, but later to be demonstrated invalid. Last winter just after my last committee meeting, the original lab working on MFP published data stating that they have been working with a protein with an incorrect translation start site. In more
lamen’s laymen’s terms, it meant the protein I had originally purified was wrong; the protein was about 15 amino acids too long. So since then, I’ve been trying to get this protein re-established and purified using their new protocol so I can redo all my old biochemistry, and finally move forward with the new biochemistry I had proposed to do. Well, it’s been nearly a year, and now I think I finally have some hope of purifying it.
- Issues that I’ve had to address include:
- Poor expression (to the point I can barely see my protein above background cellular proteins)
- Inability of affinity tag to stick to affinity resin (or any resin for that matter, ion exchange included)
- Poor plasmid maintenance after an overnight culture (and have nothing left to grow the next day)
Since then, I’ve recently introduced a new selection marker that’s far less likely to be ignored that now seems to be giving me appreciable yields of expression in a rich, defined media (instead of our typical undefined media). So right now, I’m ready to crack open all my cells and see just how soluble my protein is. If it’s well-behaved in solution, then it’s on to the FPLC and see what I can get this sucker to stick to!
I also need to submit my construct for DNA sequencing; I really want to make sure that MFP’s coding sequence is pristine. No gaffes this time! I have to sequence a bunch of my selected mutants that have passed muster so far. Up to nearly twenty candidates and I need to get some sequence back to see what kind of mutations (if any) I am getting in MFP.
Additionally, since the “correct” translational start site is supposedly published now (nearly a decade since the lab originally identified the protein in the organismal genome), we’re not entirely convinced ourselves to run headlong into this and just trust it. I did get kind of screwed by trusting the original research in the first place, no? So, I had vocalized my concerns with this being an accurate piece of data, being as how they minimal upstream sequence to incorporate any endogenous promoter sequence. Well, that came back to bite me in the rear today; my advisor would like me to affinity tag my protein with an endogenous promoter sequence and get this guy purified and sequenced by Edman degradation to verify the start site. I don’t mind cloning, but I do sort of loathe designing primers for PCR. So that’s my new side-project to verify everything is kosher, so to speak.
The genetic selection library is coming along nicely, but the undergraduate who is working on the screening is a different story. I’ll save that for another night, so I don’t unnecessarily rip on her. She does a good job, don’t get me wrong! She just tends to miss some crucial steps on the way…or a lot…yeeeeeeeah.