I am trying to produce Taq. This should be fairly straight forward, but something is going wrong, and I would appreciate some help troubleshooting what could be going wrong.
I have the pAKTaq plasmid from addgene (https://www.addgene.org/25712/), which is based on pTTQ18, with a Tac promoter and Amp resistance. I transformed this into BL21(DE3) cells, or at least I think so. The tubes were labeled only BL21, but my labmate assured me they were DE3. In any case, from what I understand this shouldn't matter for Tac promoter-driven expression. I picked colonies, grew a 500 mL culture, and followed a heat and ammonium sulfate precipitation-based prep (http://www.bio-protocol.org/e136). Checking fractions on a gel after this showed no protein. I went back and picked another colony, and tried to optimise expression of both this and the original colony (IPTG conc and expression time); no protein expression. The cells are growing in 2xYT/Amp, so I imagine they must have the plasmid. I checked the plasmid is correct by restriction digests. IPTG is being added at OD of around 0.3, and I've tried between 0.04 and 1 mM between 3 hours and overnight. If anybody can point out some obvious thing I've done wrong I would be very grateful. This has taken up far too much time as it is...
Hi Seino,
Nothing obvious comes to mind. Just to understand, when you say, "no protein expression" does it mean no band on the gel at the expected MW or the expression is not so good? If it is the former, I would think there is something intrinsically wrong. In that case I would suggest performing a miniprep and send the plasmid for sequencing. If it is the latter, I would try to optimise the expression conditions more. May be try LB media, go to OD 0.6-0.8 before inducing. Reduce the temperature to ~18C for expression (i.e after adding IPTG) ...
Thanks Sujay. It is indeed absolutely no band at the expected weight. With IPTG and uninduced control were identical. Sequencing may indeed be the only reasonable next step, or subcloning into another vector. I just want my Taq now so I can do the more interesting work...
Try using DH5alpha cells for expression. If I remember correctly I tried using BL21(DE3) once for Taq expression and also got no protein. The standard protocol in my lab uses DH5a and with this I have no problem of expressing Taq [although I'm not quite sure what plasmid we use]. A quick internet search recommended DH5a as well.
Thanks for the suggestion Sascha. It sounds a little counter-intuitive, but I'll give it a try.
(update) I tried expressing from DH5alpha, but still see no protein band on induction (overnight 0.5 mM IPTG from OD 0.3). I'm trying again now with a bunch more colonies from the plate I first streaked when I received the plasmid. Maybe I was just very unlucky in my colony choice the first time around...
Dear Seino,
I was purifying both, Taq and Phusion polymerases without any problems. Please overexpress protein in BL21(DE3) pLys but make a fresh stock of them. From my experience there is really hard to see protein on the gel. Overexpression yield is quite low. Please check the protocol attached to this post and widely aviable in the internet. I hope this will help,
Best,
Dawid
Hi Dawid. Thanks for the suggestions and protocol. The plasmid mentioned in that protocol is different from the one I have (pET28 background rather than pTTQ18), and I'm slowly coming to the conclusion that my plasmid is the problem. I have another on the way, in pET28 background, so hopefully that will solve my problems.
A follow up question about the actual purification prep: have you (or anybody else that reads this) tried the ethanol precipitation-based prep attached? The authors claim it is easier that ammonium sulfate, doesn't require dialysis to remove the salts, and carries over much less nucleic acids.
No, I haven't tried that you mentioned. It is true that Ammonium Sulphate precipitation protocol is quite complex but works really fine.
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