Nothing out of the ordinary there.
You can try to induce overnight (for 20 hours or so) at 15C. And you can
try to reduce the concentration of IPTG if excess protein is going to
inclusion bodies anyways. Collect whatever protein is soluble and ignore
your inclusion bodies. The His tags allow you
Several suggestions:
Try tagging with a solubility-enhancing tag
like GST or NusA.
If using a pET vector system, try "leaky"
_expression_. The T7 promoter is not well-repressed (except in pLysS
systems) and you can get low but very significant levels of _expression_
without induction.
try JM109(DE3) instead of BL21(DE3), combined with low temperature induction -
for several proteins this has yielded us soluble protein instead of inclusion
bodies. I think this is because JM109(DE3) grows a bit slower.
Mark J van Raaij
mvr...@ibmb.csic.es
I have found that if you give a cold shock ( 4 degrees for 30 mins-1 hr)
before low temperature induction it helps to keep proteins
soluble.
Ivan
English et al. Recombinant and in vitro expression systems for hydrogenases:
new frontiers in basic and applied studies for biological and synthetic H2
production. Dalton Trans. (2009) (45) pp. 9970-8
I've never tried to express a hydrogenase, but I've read that it's very
hard. Do you have all
Dear all,
I am trying to purify a metalloprotein (a hydrogenase) using affinity
chromatography.
I have produced two tagged versions of the enzyme: one with an N-terminal 6x
histidine affinity tag, and the other with a C-terminal 6x his-tag. The tagged
proteins are both tied to an
Buz,
How big is the protein? How many metal binding sites are there? Do you know
what metal it should bind? What is your media?
I am assuming it is a zinc binding protein, some zinc binding protein will not
fold without enough zinc in the media. Perhaps you should not rule out
refolding.
ray
Buz,
Have you tried putting on a solubility tag, such as GST?
I've found some times using M9 to grow my proteins in can give more soluble
protein, although yields are usually lower.
Best,
Peter
