Dear Bob (and Bob)
In the same line of thoughts, I would suggest you to try NOT to mess around
with inclusion bodies. My experience with them is mostly negative, if I can
remember correctly what happened many years ago. So, 5 or 6 years ago we
switched to fusion proteins "GST-our protein", obtainable with the pGEX
vectors from Pharmacia, and since then I would not try anything else:
1) The fusion gene is under the control of the tac promoter, which allows
chemically inducible, high-level expression of proteins. There is no
expression until you add the IPTG, and the latter is added for just 2-4
hours, so that even toxic proteins can be produced at high level.
2) The proteins are soluble.
3) The cells are sonicated and the total lysate is loaded onto a
Glutathione-Sepharose column that will retain the GST, the other hundreds
and thousands of proteins will be gone with the flow through, and the
consecutive washes. The SDS-PAGE gels after elution (with reduced
Glutathione) are always consistent, NOTHING BUT YOUR FUSION PROTEIN (unlike
the INCLUSION BODY extracted proteins). The column may be reused many times
for different proteins.
4) The fusion proteins are always excellent to raise antibodies in rabbits,
we have done this for several proteins, and all of them were immunogenic in
a single rabbit. The animal is not mistreated, the protein is injected in a
PBS solution (not a 4M urea!). The anti-GST component of the polyclonal
serum does not result in non-specific binding to mammalian proteins.
5) In additon to antibody production, you can also perform functional
studies with the GST-X protein, using affinity chromatography with a very
simple setup: you can attach (or not elute from the start) your GST-X
protein on the same Glutathione Sepharose Column, and pass a solution of
whatever nature (protein or ligand, total nuclear extract, or candidate
drugs), and only those reacting with the GST-X will be retained (this could
be useful to study, say, dimer formation of novel transcription factors).
6) Usually the GST-X proteins retain the function of the X protein (it could
be though that we were lucky).
7) Once you construct the first expression clone, the process can be
repeated for other proteins, without additional "optimization".
Disclaimer: I am not in any way related to Pharmacia, besides you may be
able to buy similar vectors from their competitors, or ask a colleague .
Best regards,
wagner
-----Original Message-----
From: Association of Biomolecular Resource Facilities
[mailto:abrf-request@aecom.yu.edu]On Behalf Of Bob Keefe
Sent: Thursday, September 14, 2000 9:06 AM
To: Recipients of ABRF List
Subject: Re: Inclusion body
Dear Bob,
Have you made any attempt to grow your E. coli (that are recombinantly
expressing the protein you're studying) in different media to eliminate the
presence of inclusion bodies? In the reference "Overcoming Inclusion Body
Formation in a High-level Expression System" (J. T. Moore et al., (1993)
Protein Expression Purif., pp 160-163), the authors describe an "enriched"
growth medium they tried/developed to over-express their enzyme (using the
pET system from Novagen) in an active form with good results. Previous to
doing this, their enzyme was expressed in an inactive form in inclusion
bodies. Of course, if all you need your protein for is to generate
polyclonals, activity may not be an issue here. Just the same, this might
be an alternative approach you might want to consider.
Best of luck,
Bob Keefe
Genomics Core Facility
Wadsworth Center/NYS DOH
At 01:43 PM 9/13/2000 -0400, you wrote:
>Hi,
>
>Need a good way to prepare the protein from the inclusion body of the E.
>coli for raising polyclonal antibody. Thanks.
>
>Bob
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