T7 Expression System - (Dec/31/2011 )

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 however there is no T7 terminator present as far as I know. 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 primarily 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-R/Tet-R genes and thus make it somewhat harder to purify the gene of interest?

I plan to assay a mutant protein against it's wild-type form and compare the activities thus I require a high purity as I need to know the concentrations of each to be able to compare the result from the bioluminescence assay.


Also do phagemids simply contain bacterial promoters too so that Amp-R/Tet-R can be expressed?


Is there any other way I can express and purify this protein (it's an adenylate kinase) in vivo, rather then simply using the T7 in-vitro system. Thanks,

TJ Cooper

Phagemids contain promoters for the Tet/Amp cassette, but they will not express from the T7 promoter unless there is read through. You could eliminate this by orienting your cassette in the opposite orientation, or, as you suggest, adding a terminator. An alternative would be to use an in vitro protein production system such as the PURE system (NEB), which would likely yield a purer product. You could also consider tagging the protein with a 6HIS or chitin binding protein and doing affinity purification. The NEB Impact system can produce pure native protein by cleavage of a chitin binding domain.

phage434 on Sun Jan 1 00:27:10 2012 said:

Thank you for your reply however I am limited to using the system at hand by my lecturers. This means I am unable to introduce tags for affinity columns, using the NEB system or introducing a terminator and must utilise the system at hand which is far from ideal.

EDIT: As i'm dealing with an adenylate kinase I thought about simply inducing expression via the T7 promoter then using blue-sephadex in affinity purification which seemingly binds to dinucleotide folds in adenylate kinases with a high affinity and then eluting with nucleotides or Ap5A. Does that seem viable?

just out of curiosity ...is there any rational behind using a heat-inducible version of the T7 RNAP on a plasmid rather than using a simple DE3 lysogen strain like BL21(DE3)?

Many thanks in advance!

Regards,
p

pDNA on Sun Jan 1 10:43:34 2012 said:

I fully agree with you and if given the chance I would have used such a strain - unfortunately this is a guided first year university project and I am restricted to using the systems at hand.

okay - i thought it is a special system intended for the labeling of proteins!

Regards,
p