Desperately Seeking pRSET
QIAGEN at kaiwan.com
Fri Jul 7 11:21:05 EST 1995
In Message-ID: <3sl4e4$k0o at newsbf02.news.aol.com>
Nigel Walker (squirenige at aol.com (SquireNige)) wrote:
>... so consider whether a c-terminal fusion system may be more to your needs.
>Qiagen supply suitable vectors that generate C-terminal his tags.This will
>allow purification of full length proteins only.
>Also people tell me this system tends to produce insoluble proteins all
>the time. Both proteins we have expressed are totally insoluble, so also
>consider if this will be a problem for you. Pharmacia do a GST-fusion
>system that helps to keep things soluble as do Qiagen with a DHFR His-tag
We would like to correct a misconception. The DHFR-fusion does not keep
proteins soluble. DHFR is a highly expressed protein which is normally
insoluble in under 1M urea. It is often fused to short sequences, or poorly
expressed proteins to increase the expression level.
The QIAexpress pQE vectors use a bacteriophage T5 promoter which gives the
same high expression levels as phage T7 promoters. Expressed proteins can
become insoluble for several reasons, one of which is too high or too rapid
expression, so changing to a weaker promoter, or lowering the growth
temperature can help. Reducing the amount of IPTG used for induction also
reduces expression in the pQE system, which has two lac operator sequences
associated with the promoter, for tighter regulation of expression.
Unfortunately, solubility must often be traded off against expression level
Some proteins which are very hydrophobic, need certain cofactors for
solubility, or require other proteins for correct folding, will often be
insoluble. You can sometimes keep them soluble by modifying the expression
conditions Ñ media, host, temperature, length and time of induction Ñ but
it is highly dependent on the nature of the protein. Some proteins can be
kept soluble by secreting them into the media. GST-fusions are often more
soluble than the native protein, but the protein can precipitate once the
GST is removed (if removal is necessary).
Once a protein has precipitated, it can generally only be resolubilized by
denaturation, although some proteins can be solubilized in detergents like
CHAPS, without denaturation. The advantage of the QIAGEN 6xHis Tag/Ni-NTA
system is that the protein can be purified after solubilization in high
levels of denaturants like urea or guanidine hydrochloride and then
in solution, or while still bound to the resin, if desired. Proteins
solubilized in 1% CHAPS can also be purified on Ni-NTA resin.
The QIAGEN Technical Service Department has extensive references available
for purification of soluble and insoluble 6xHis-tagged proteins.
QIAGEN Technical Service Department
Temporary Email Address qiagen at kaiwan.com
More information about the Methods