Dear Iva,
Feel free to email me rather than posting - unless you feel that this
eschange might benefit the group in general. I am a crystallographer myself
and your problems sound quite familiar. Here's what I suspect may be
happening, based on what you say:
> When I purify the fusion with FPLC- monoQ something strange is happening.
> After SDS-PAGE of all fractions it appears that my protein is eluting at 2
> different salt concentrations- at around 200 mM and 500 mM NaCl. Does that
> mean that some aggregates or complexes with other proteins are being
> formed???
Not necessarily so. 500 mM is quite a lot of salt. Have you considered the
possibility that your protein binds to oligos or long DNA ? 500 mM is about
where some DNA starts to elute off the Q columns, and in many cases in the
past we have seen co-elution of 'stuck' proteins. Check your OD260/280
ratio, especially that of the higher-salt fraction - does it look like
protein only or does it have abnormally high 260 ? Aggregates can stay that
far into but that's somewhat unusual.
> I have done protease cleavage for removing MBP only on protein
> after amylose column before MonoQ. Now I am doing cleavage on protein
> fraction 200 mM NaCl from MonoQ. And I will run gel filtration. BUT my
> question is since I have done cleavage in presence of Tween 20 and
Triton-X
> 100 (0.01 and 0.1%) isn't that going to disrupt any aggregation or
> interaction with other proteins???
Unfortunately, Tween and Triton are tricky detergents. They may or may not
help you, depending on the degree of severity of the aggregation. This kind
of detergents is considered by many to be a last-resort option, have you
tried milder stuff such as BOG or dodecyl maltoside, etc. ? Did you try
adding detergents on lysis, rather than on cleavage (by then it may be too
late!).
>And if that (agregation or interaction)
> is the problem cloning another protease clavage site probably won't help.
> What will?
Seems like you are running into the area of 'my protein sucks, it aggregates
and sticks to stuff' kind of problems. There is no generic solutiuon,
however to name a few - people have tried to use detergents during lysis,
induction at lower temperature, co-expression with various complex-forming
partners, and many other tricks. Can you tell in a bit more detail what is
this protein like - is it a DNA-binder, is it supposed to bind other
proteins, is it heterologously expressed, does it have native disulphides or
suchlike, etc. Have similar proteins been expressed and crystallized ? How ?
Good luck. Protein purification is tough :)
Artem Evdokimov
