Assuming you can get the protein in solution, it should purify really
easily on a hydrophobic interaction column. When I was on sabbatical
leave at the NIH I helped a colleague purify a really hydrophobic,
aggregation-prone protein this way, and it was tantamount to affinity
purification. We used butylsepharose, which is the weakest of the HIC
columns. Worked like a charm.
The trick will probably be getting your protein soluble and
aggregation-free. Small amounts of detergents? urea? GuCl?
Cheers,
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [email protected]
On 2/27/2014 3:45 PM, Allan Pang wrote:
Hi all,
Any of you got some experience on purifying (and crystallizing)
VIL-rich proteins (actually, more particularly isoleucine rich). Tried
to purify my protein, but majority (if not all) of the proteins reside
in the inclusion bodies. Induction is at low temperature, overnight
and tried doing urea treatment, but oddly the his-tagged protein
'loses' it's ability to bind to nickel column. When unbound protein of
interest ran in size exclusion column, it's eluting at void volume.
Also tried with working on a different Bl21 strain (Rosetta and RIL),
out of luck.
Ile (I) 20 12.0%
Val (V) 14 8.4%
Leu (L) 13 7.8%
Asn (N) 12 7.2%
Glu (E) 12 7.2%
Ser (S) 11 6.6%
Lys (K) 11 6.6%
Any suggestion would be nice, or point to direction where other
successfully purify and crystallize sort of similar protein. BLAST
does not give me a close homologue of the protein that I can work
with, so no information on possible domains.
Thanks
Allan
