GST-fusion proteins and bioactivity

Dominic Voon dvoon at pleaseremove.cyllene.uwa.edu.au
Mon Nov 22 00:07:44 EST 1999


Hi all,
	I've been having problems with GST-fusion proteins which don't
seem to be bioactive. The fusion protein itself, when visualised on a
SDS-PAGE gel appear as a very nice clean band with very little or no
GST-alone degradation products. I had a thorough search through the
newsgroup archive but have found very little information concerning the
bioactivity of GST-fusion protein. In this case, we believe the protein of
interest has RNA binding activity and have in the past shown that the
GST-fusion does demonstrate this activity. The protocol has worked once in
my hands (ironically, the first time I did it) and has since then failed
to produce functional protein. I have tested various aspect of the
protocol, such as the lysis condition (we use a combination of
TEG+lysozyme; TritonX-100; and passage through high-gauge needles), the
amount of DTT, protease inhibitor etc. The previous person had not
encounter this problem (as far as I can tell) but she did have problems
with protein degradation. Could my problem  be due to too high a
concentration of protease inhibitors ? I am currently using 0.5 mM PMSF
(with regular replenshment), 1ug/ul aprotinin, leupeptin and 1 mM DTT.
As I've mentioned, the protein preps themselves are pretty clean and I do
get very good yield (~1-5 mg per 1 L prep).
	Someone brought to my attention the issue of the promoter leakage
in the pGEX4T construct which we use. Because I am currently growing up my
bugs in straight LB and without inhibitors like maltose, the low level of
basal expression in the bugs may lead to a selection of clones which has
large number of protein inclusion bodies (which may explain the high
yield). What I am uncertain of is what happens to the GST-protein once
they are 'dumped' into the inclusion bodies ?
	Finally, assuming that my problem of low/no bioactivity is in fact
due to some form of denaturation, is there a way of rescueing them ? I
would appreciate if someone can point me to a good renaturation/refolding
protocol.

Your help, as you can imagine, will be much appreciated :-),
dominic

NB: Please note that I have added a "Please remove"  tag to my email
address to avoid spammer.




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