Normally Monday’s aren’t such a struggle. However, today was a two pot of coffee kind of day. Yawn. I think most of the water I drank today came from coffee. Probably not the best thing.
On the research side of things: I’ve been working on producing this one protein for maybe a year and a half now (closer to two years, but saying a year and a half reduces my anxiety) and I’ve been able to produce it since July. Yay! But, I’ve had enormous purification issues. Basically, this protein comes from a baculovirus expression system (don’t even get me started on the manufacture’s protocol for that) in insect cells. The fun part is that the media for the insect cells contains what I have deduced to be some sort of 20-30k polyamine polymer. This polymer interferes with His-tag immobilization by stripping the Ni-NTA column of all it’s nickel. Yeah, real fun to see your nice blue column go white.
The typical way of dealing with this polymer is to do a buffer exchange with an anion or cation resin. Well, of course, my protein is a heterodimer and of course it is sensitive to these resins. I loose the dimer after the column. Monomers for days. Last week however, I found that using a tangential flow filtration (TFF) system I could remove this polymer. Or so it seems.
As a side note, for those who haven’t used a TFF system before it really is great when dealing with large volumes. I do these protein extractions on the liter scale. But don’t believe the manual. This 51 page manual starts with describing how “old” dialysis is and how you can loose your sample by dropping it on the floor. Then states that TFF is so much easier to use. Just read our 51 pages on how easy. Spoiler, it’s not so easy at first.
But heck, now that I’ve done it a few times and was able to get pure protein (not using a Ni-NTA column for purification. That story is for another day.) I should be to do it again, right?
So last week I set-up this buffer exchange and start concentrating the sample, then do another buffer exchange and I leave for the night after calculating how much buffer I need to last the few hours. In the morning the filtrate has gone cloudy…it’s never been cloudy.
Sure enough, I elute the protein off the column and that is cloudy too. Strange. But like any good solider I push though and purify the protein. Then a Bradford tells me I have no protein (or very low) in my elutions. Time for a coomassie gel. I’m not too hopeful at this point and it’s a Friday and I’ll have to come in on the weekend to destain and take an image. No problem, typically happens. But this last weekend found me drenched with house chores and it just didn’t happen.
So Monday comes. Lovely Monday. Two pot of coffee Monday.
The coomassie shows purified protein though. Huh, okay I’ll take it. There was some strange precipitate in the elution fractions after starting a new dialysis (have to remove the low pH buffer used for elution).
That got me thinking back to the mysterious cloudy filtrate. I bet the polyamine polymer crashes out of solution at a higher pH. You see, the insect culture media is around pH 6-6.5. My buffer was at pH 8 so it crashed out then (I think the first time I did this my buffer was at pH 7.4) and then the left over polymer went back into solution during my pH 3.5 elutions off the resin and subsequently crashed out when being dialyzed into water.
So that was my conclusion to the cloudy Monday saga.
On a side note, while running a Western to verify all this, I took a walk around Boston during an incubation step. I’ve never been a big city kind of guy, but there is something quite romantic about the old brownstones on a picturesque fall night in Boston. The city has grown on me over the past 3 years.