Hello Rafael,
I would give one more by reducing the temperature for the expression. I am
assuming that you did the expression at 37 oC. I would start expression at 30
oC and reduce it further after induction to 25 oC or so and let it grow longer.
But refolding the protein is also possible. The paper you attached is one way
of doing it. The second method is step-wise dialysis and the link to this
protocol reported in an article is below. You can try try both methods and
check for yield. Overall, I prefer smaller tags such as His-tag or GB1-tag just
because you do not lose 50 % or more of your expressed protein due to a large
tag. Good luck! Thomson, C. A.; Olson, M.; Jackson, L. M.; Schrader, J. W.
Asimplified method for the efficient refolding and purification ofrecombinant
human GM-CSF. PLoS One 2012, 7, No. e49891
Best wishes,
Smita
Professor of ChemistryAdjunct Professor, Biochemistry & Molecular
BiologyOklahoma State University, Stillwater, OK 74078, USA
On Tuesday, March 11, 2025 at 06:18:46 AM CDT, Rafael Marques
<[email protected]> wrote:
Hello folks,
I have been trying to purify a GST-tagged protein for a while but the results
are far below the expected. The majority of my sample is in the pellet fraction
and the yield of my expression is very good. This is known because the protein
was identified both by Mass Spectrometry and Western-blot. I believe my protein
is laying inside inclusion bodies. I have tried to diminish the IPTG
concentration, express it for a shorter time and also different E.coli strains
but the results are still the same. As a last resort, I was thinking about
refolding. I found this paper (attached), from 2004, but I was wondering if
someone who has experience regarding protein refolding could shed some light on
this topic, principally if there are more updated protocols.
Best wishes
P.S. His-tag did not improve my results too
______________________________________________________
Rafael Marques da Silva
PhD Student – Structural Biology
University of Leicester
Mestre em Física Biomolecular
Universidade de São Paulo
Bacharel em Ciências BiológicasUniversidade Federal de São Carlos
phone: +44 07861 273773
"A sorte acompanha uma mente bem
treinada"________________________________________________
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