pGEX (GST) expression system
dalmiabk at phibred.com
Wed Mar 8 16:45:06 EST 1995
i have had very good success with the gst-fusion system for protein
expression, followed by purification on gsh-agarose, followed by thrombin
cleavage and removal. currently i am expressing couple proteins with this
system that refuse to be produced in a soluble form even with all the
true and tried conditions (low temp., low IPTG, BL21 etc.). so i went
ahead and OPTIMIZED for inclusion body production and from a 1 liter
culture purified tons of insoluble fusion protein by repeatedly washing
with 2.5 M urea. a part of this would go for antibody production. but now
i need my protein WITHOUT the GST portion, and in a soluble form.
has anyone tried sarkosyl solubilization with purified IBs? and would
thrombin cleave the fusion protein in the presence of 1.5% sarkosyl? if
not, what other techniques do you recommend? i am in the process of
solubilizing the IBs in 6-8 M urea and trying to refold.
for the sarkosyl solubilization DURING cell-lysis, and more developments
since the Farngioni and Neel publication?
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