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T7 Expression System

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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T7 Expression System

Postby TjCooper » Sat Dec 31, 2011 5:46 pm

Good evening,

I am currently a first year molecular biology student at university. I just have a question and I hope that it's okay to ask here. I currently have a simple expression system setup within E.Coli K12 strains, using a phagemid with Tet-R/Amp-R genes, and my gene of interest inserted downstream of the T7 promoter. I plan to introduce a heat-inducible plasmid containing the gene for the T7 RNA polymerase under the control of an E.Coli promoter into the cells, and induce it in the presence of labelled amino acids, and Rifampicin to inhibit endogenous RNA polymerases in the hope of translating the gene of interest, labelled, and easily purified. My question is - knowing that the T7 RNA polymerase can be highly processive, is it likely to also transcribe the Amp/Tet genes and thus make it somewhat harder to purify the gene of interest? Thanks,

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Postby JackBean » Tue Jan 03, 2012 7:46 am

Trust me, if you had to purify your protein of interest only from three proteins, you would be lucky bastard :lol: However, the resistance genes cannot have T7 promoter, otherwise the bacteria would be resistant only if your protein would be expressed. Thus they are expressed constitutively. Thus if the T7 polymerase would be able to transcribe them, it would transcribe also other endogenous genes.
But you do not have to worry, since it should be extremely specific.
Anyway, you do not use any tag?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: T7 Expression System

Postby TjCooper » Tue Jan 03, 2012 11:55 am

:lol: Thanks for your reply! Unfortunately I have been restricted in using the system at hand which includes no usable purification tag. I guess I will run it through a blue-sephadex affinity column (as it's a putative adenylate kinase) and see where it goes from there!
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Postby JackBean » Tue Jan 03, 2012 1:10 pm

you will extract lots of endogenous proteins anyway, because they are present in the cells, although transcription will be inhibited. I would recommend ionex first and then you may try the blue-gel and/or some gel permeation chromatography
http://www.biolib.cz/en/main/

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Re: T7 Expression System

Postby TjCooper » Tue Jan 03, 2012 1:22 pm

Yeah the endogenous proteins are a big problem. Alternatively I could do a standard maxi-prep plasmid extraction, and use it in a in-vitro transcription/translation system with T7 RNAP then run the blue-sephadex affinity column but also wanted to establish an in-vivo method to try.

I've noticed here:

http://www.sciencedirect.com/science/ar ... 9383806243

The use of blue-sephadex, Ap5A elution and a second G-100 sephadex separation yield homogenous adenylate kinase in E.Coli anyway (the putative gene i'm dealing with show's a high degree of similarity in sequence to E.Coli AK). However this would not solve the problem of the endogenous E.Coli adenylate kinase so i'm favour of using an in-vivo method.

Thanks for your help!
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Postby JackBean » Tue Jan 03, 2012 1:59 pm

yep, it probably will be problem, but if you induce it a lot, you should have excess of your AK over the endogenous one.
http://www.biolib.cz/en/main/

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