Over-expression using maltose-binding protein

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Thu Oct 4 22:20:55 EST 2001


Ok. Let's see...

You have MBP, with a pI of about 5.2 and size of about 42 kDa and protein X
with pI of 9.5 and m.w. 30 kDa. First thing I'd do is to pass the mixture
through an amylose column and see how much MBP (and uncleaved fusion, if
any) will remain afterwards. Amylose affinity works well in 300 mM salt at
reasonable pH (say, 8). Might want to go even higher in salt  - I *think*
that 500 mM NaCl is still OK.

Certainly, this will not remove all MBP but, hopefully, will make the ratio
favorable towards further purification by generic methods. With pI of 9.5
(!) the protein should be easily purifieable on CM resins, you can probably
load it in quite high salt so nothing else will bind at all. Play with the
pH of the buffer, too. Add some (20% ?) ethylene glycol or low m.w. PEG to
your buffers, see if this helps.

Will it elute off CM ? No idea ! You say that it is being lost. Perhaps your
carrier resin is acting as a hydrophobic matrix ? Perhaps you can succeed
with using very weak HIC matrices, something as short and punk as Ethyl (or
even methyl HIC - do they even make that ?). Elution with ethylene glycol or
glycerol comes to mind, since simply lowering the salt may or may not work.

Shot in the dark: add some octasulphonyl sucrose to your solutions. It may
help keep your protein in solution. Or, it may kill it. Can't say w/o
trying.

Hope this helps !

A.G.E.


"Arsenio Villarejo" <arsenio.villarejo at plantphys.umu.se> wrote in message
news:a04310108b7e243d742f8@[130.239.110.34]...
> Hi,
>
> I'm trying to over-express a protein using the
> maltose-binding protein system. My protein is quite hydrophobic and
> has an isoelectric point close to 9.5. After cutting the fusion
> protein with Xa factor, I tried to purify the cleaved protein by ion
> exchange chromatography. At this point, I lost my protein because it
> binds to everything (dialisis membranes, centricon membranes,....).
> So, I should keep a relative high salt concentration in the mixture
> to avoid any precipitation of my protein.
>
> What kind of chromatography technique can I used for
> separating the maltose binding protein from my protein (with ca. 30
> kDa)??.
>
> Any help or new idea will be wellcome.
>
> Thanks a lot in advance.
>
> Arsenio Villarejo
> --
> Arsenio Villarejo                            Phone:  +46 90 7867648
> Department of Plant Physiology               Fax:    +46 90 7866676
> Umea University
> S-901 87 Umea, Sweden     E-mail: Arsenio.villarejo at plantphys.umu.se
>
> ---





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