insoluble proteins
govind kunduri
Tuesday, 04 September 2007 14:18 UTC
hello
Iam working on a RNA dependent RNA polymerase(RdRp) of a plant virus.For in vitro studies i have cloned RdRp in bacterial expression vectors such as PRSET-A, and pET22b which have his-tag.But all the protein was found in insoluble fraction, after trials of low temperature, low IPTG,2% ethanaol had now improvement on solubility.GST taged protein was found in soluble fraction but it is highly prone to proteolysis(which was overcome by 2mM EDTA) and is also not binding to GSH resign(according to novagen manufacturares protocol).
please suggest how to get soluble protein with His tag proteins and how to purify GST-taged protein which is not binding to resin
Updated 04 September 2007 14:21 UTC
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Replies
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Hi Govind,
The following paper, recently published in BMC Biotechnology, might be of interest to you: Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli.
Alternatively, this Nature Protocol may be of help: Single protein production (SPP) system in Escherichia coli. Volume 2 of Nature Protocols is freely available until the end of October, so you should have unrestricted access to this article until then. If you do try out this Protocol, we would encourage you to leave feedback for the authors and other users by posting a comment.
Hope this helps – and good luck!
Dorothy -
hi govind, my self manish goyal PhD student , i faced the similar problem as u found , if u get any solution , can u help me.
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