T7 Expression System - (Dec/31/2011 )
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:
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.
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:
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
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