ffrank at rz.uni-potsdam.de
Fri Aug 16 04:14:32 EST 2002
mukherji at tifr.res.in (Sulakshana Mukherjee) schrieb:
> Dear All,
> I am presently working on HBx protein found in HepatitisB virus. The
> protein has been cloned and over-expressed in E.coli. It is a His-tagged
> protein. It has been found that the protein goes in to the inclusion
> bodies when overexpressed. It has been reported that the protein has a
> rapid turnover.
> I wanted to know what does rapid turn over mean?
That means that it has a short lifetime in the cells, it is continuosly
depleted and re-synthesized. This may be because it is specifically
degraded or because it is rather unstable and tends to misfold (and be
degraded by unspecific cellular mechanisms).
> And also wanted to have a
> protocol for the purification of HBx. I am basically an inorganic chemist.
You should consult a book on protein purification. Perhaps you'll find
one that covers individual purification steps, but not how to find a
strategy, so I'll give you some ideas:
If it is His-tagged and in inclusion bodies, you first have to denature
the inclusion bodies and renature the protein. Then the the first
purification step would be affinity chromatography (usually Ni-chelate
column). There are several possibilites for the order of the steps,
either 1. renaturation 2. affinity chr., or (rather) 1. load on the
column, 2a. elute, 3a. renature or 2b. go to native buffer on the column
and 3b. elute native protein.
For some purposes this might be enough (run an SDS gel and read about
the purity requirements for the application you want to do). If you need
a higher purity, which will often be the case, you will probably do more
chromatography. To chose which type is a question of experience
(i.e. alchemy), and ideally of considering the properties of the protein
and the impurities. Possible choices are ion exchange chromatography
(chose buffer conditions and anion or cation exchange based on the
calculated pI of your protein), hydrophobic interaction chromatography
or gel filtration.
If you didn't renature on the His-column, but rather by diluting
afterwards, you usually need to concentrate the protein. For this, you
should consider old-fashioned ammonium sulfate precipitation, because if
you do fractionated precipitation, this is a purification step, too. But
keep in mind that there might be soluble, but still non-native protein
in your preparation. This might precipitate at different saturation of
Ammonium sulfate, but is indistinguishible on an SDS-PAGE.
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