my protein His-Sumo-insert (Theoretical pI: 5.84), is a plant protein, and it's getting expressed in ecoli, but everything was going into pellet fraction. i have tried changing cells (BL21, Rosetta, Chaperone cells), changing buffers (HEPES, phosphate), and changing pH(6.8-8). Last but not least, I have added 0.1 % SDS to my lysis buffer, and my protein is coming little bit in the sup fraction, but now it's not binding to my nickel beads. I have been stuck with this problem for two years, so please help. I can't denature since we do structural studies.
my lysis buffer composition - Tris 50mM, lysozyme, 0.5M NaCl, 1mM DTT, 1mM PMSF, 1% tween 20, 0.1% SDS, 10mM MgCl2, pH = 7.4.
we don't have insect cell or yeast facilities.
Dear Arpita Das
i thnk that the main problem is that you protein is unfolded and it tends to aggregate, in this way the histidine tag is not exposed but buried into the aggregate and the protein is not able to bind to the IMAC.
I understand that SDS can solve a little this problem, but it is also denaturing and it produce big size micelles that are not compatible at all with structural biology.
Generally for membrane protein other less strong but more expensive detergents as DM, DDM or OG are used in the lisis buffer and purification buffers.
is you protein a membrane protein or is just a soluble plant protein carryng post transaltional modification as many S-S bonds, glycosilations that are not performed by E.coli?
because in the first case (membrane protein) the use of a mild detergent could be a solution, but in the second case, i think that is it better if you leave e.coli and move in a mammalian expression sistem (ae expi293, CHO)
there are some chinese companies as biointron or Genescript that offer mamalian cell expression service with reasonable price. of course in this way you can only produce unlabelled protein (not 15N or 15N /13C) and it depends wich is your structural biology approach, NMR? X-ray? Cryo-EM?
best
Manuele
you might consider using an alternative affinity chromatography method.
@ Sebastian
- yes i have tried as low as 12-degree temp, not much difference. tried to check the expression right from 3 hours at 12 degree, its going into pellet.
- no my protein is not codon optimized, I have cloned it from Arabidopsis.
- I did use urea, but since we are planing for structural studies, refolding is not preferred.
- no purifying doings separately is not my agenda. i did not get this question, can you please explain, I want to get the whole protein intact.
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