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.