Hi,
If you are plagued by ‘generic’
proteolysis, it is not likely that changing buffers from TRIS to phosphate will
help reduce the breakdown. You may want to ask yourself several key questions
regarding the breakdown:
- is
it proteolysis or abortive translation?
- is
it happening during expression or post-lysis?
- is
it exoproteolysis or endo- (or both)? Which terminus is more affected?
If you have abortive translation or
alternate transcription/translation starts, you should examine the DNA/RNA
sequence of your gene closely – sometimes you can tell (e.g. beginning of
the gene is peppered with methionines and S-D sequences)
If you have proteolysis during expression,
it sometimes can be alleviated or even eliminated by changing expression
conditions (temperature and richness of media seem to be key – this
summer we had exactly this kind of a situation where expression at 37C for 4
hours gave more total protein than expression at 20C overnight, however the 37C
protein was significantly ‘busted up’ whereas the 20C was
essentially intact).
If you have proteolysis during cell lysis
you may be able to reduce its extent by means of at least the following (not a
complete list by any means!):
a) lyse and process the cells on ice or even in liquid nitrogen (see
some previous posts regarding mortar-and-pestle LN2 lysis). Avoid sonication or
other forms of mechanical lysis in-liquid as they all generate heat (detergent
lysis or French press may be safe alternatives)
b) make an effort to lyse the cells and complete primary extraction as
fast as possible (for example, in an extreme case you can forego the lysate
clarification step – high-flow IMAC resins can tolerate complete crude
lysate in batch mode or sometimes even on a column).
c) use protease inhibitors (can be somewhat expensive)
If you can figure out which end of the
protein is affected (or perhaps its lysis of an interdomain linker, exposed
loop, etc.) you may be able to re-design the construct to avoid this.
Presumably the fact that your protein can be extracted via the His6 tag means
that the end with the tag is intact (or at least enough of it is intact to make
purification possible). Therefore it may be interesting to switch the tag to
the other end – you may get lucky.
If you cannot afford the (expensive)
commercial protease inhibitor cocktails, you may be able to get away with using
the ‘old basics’ such as benzamidine, PMSF (AEBSF), and EDTA/EGTA
or phenantroline. Yes, you won’t be able to use IMAC together with EDTA –
but you can always do cation exchange first (protein with pI of 10!!!) in EDTA,
then polish it up with IMAC etc. If your protein is really pI 10, it should
bind to CM-sepharose in buffer with pH as high as 8, which is essentially a
guarrantee that your protein will be almost alone in the extracted material –
you wil likely see a ribosomal p10 protein that’s about pI 11 – and
that’s it. Remember that if you go this way, you should avoid using
lysozyme for cell lysis since HEWL is mighty basic itself.
Good luck,
Artem
From: CCP4 bulletin
board [mailto:[log in to unmask]] On
Behalf Of
Sent: Friday, August 22, 2008 6:16
PM
To: [log in to unmask]
Subject: [ccp4bb] protein
degradation
Hi,
This is going to be an off topic question concerning this community. I have a
protein 6XHis tagged. When retrieved from the Ni-NTA column with imidazole
found to be degraded, appears like a deep band with other bands (touching each
other below the main band) in SDS PAGE. The protein is a DNA binding (pI~ 10)
and Tris-Hcl buffer is used with 10% glycerol and 300mM NaCl. for purification.
Does Phosphate buffer do any help in stopping the degradation.
All suggestions are welcome. Thank you for your reply in advance.
Sincerely
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