10 replies to this topic

[chj]

Hi,all

I have got troubles with protein pruification recently. My protein is a little large, 900+aa, and constructed to pGEX-6p-1, expressed in Rosetta(DE3). The fusion protein exists in supernatant after lysising the cells, while exists more in precipitate. I just take the supernatant to subsequent steps. But eluting the protein from the gst column, I found the purity is very low, no matter I cleave the PrePcission on or off the column. I am sure I have wash off the undisired protein with sufficient PBS.  My protein contains 16 Cys, is it make the elution low purity?
So, could anyone help me analyse the problem and provide some suggestion about purification of Cys containing protein
Thanks so much!

chj

Edited by chj, 03 May 2010 - 08:30 AM.

[mdfenko]

you can try adding some dtt to the buffer (1-2 mM) to keep your cysteines reduced. it should prevent any contaminants from binding to your protein through disulfide bridges.

[chj]

mdfenko, on May 4 2010, 10:42 PM, said:

you can try adding some dtt to the buffer (1-2 mM) to keep your cysteines reduced. it should prevent any contaminants from binding to your protein through disulfide bridges.

thank you
I have tryed, but it had no effect
and I concern whether dtt would destroy the right disulfide bond formed in the inner of protein

[mdfenko]

chj, on May 5 2010, 12:01 AM, said:

thank you
I have tryed, but it had no effect
and I concern whether dtt would destroy the right disulfide bond formed in the inner of protein

at 1-2 mM dtt will maintain sulfhydryls but will not break disulfides.

you would need to add it before disulfide formation.

Edited by mdfenko, 05 May 2010 - 06:19 AM.

[swanny]

What are you using to wash off non-specific binders? Try adding 10% glycerol to your wash solution.

[Another Jake]

0.1-0.5% NP40 in your washes can help too.  Also, are you certain that your "impurities" are not just degradation/cleavage products of your protein.  That is VERY common in GST preps.  That can alternatively be the result of codon issues when producing a eukaryotic protein in bacteria.  We've recoded a few of our smaller constructs to deal with this.  Otherwise, we used "codon plus" bacteria which express the tRNAs needed to avoid the problem.  Both methods helped reduce the smaller bands.  That solved it almost entirely for a couple of the proteins, though not all.
Also, the DTT should be added before you lyse the cells (even before sonication).

Another alternative is to tag both termini of the protein and do a double purification.  That covers degradation issues and codon issues, though you lose yield.
If those are not the problem, consider a single-terminus tandem affinity purification.

edit:  One other alternative is to test different induction times and temperatures.  Some proteins are expressed well in a stable form in only 3 hours at 37 after induction, but some others do better at room temp for longer periods.

Edited by Another Jake, 05 May 2010 - 08:25 PM.

[chj]

mdfenko, on May 5 2010, 10:18 PM, said:

chj, on May 5 2010, 12:01 AM, said:

thank you
I have tryed, but it had no effect
and I concern whether dtt would destroy the right disulfide bond formed in the inner of protein

at 1-2 mM dtt will maintain sulfhydryls but will not break disulfides.

you would need to add it before disulfide formation.

thank you
indeed, i added dtt just when washing impurities and eluting target protein

[chj]

swanny, on May 6 2010, 08:57 AM, said:

What are you using to wash off non-specific binders? Try adding 10% glycerol to your wash solution.

pbs, tris-hcl+triton+salt,...
thank you, I will try to add glycerol to wash

[chj]

Another Jake, on May 6 2010, 12:22 PM, said:

0.1-0.5% NP40 in your washes can help too.  Also, are you certain that your "impurities" are not just degradation/cleavage products of your protein.  That is VERY common in GST preps.  That can alternatively be the result of codon issues when producing a eukaryotic protein in bacteria.  We've recoded a few of our smaller constructs to deal with this.  Otherwise, we used "codon plus" bacteria which express the tRNAs needed to avoid the problem.  Both methods helped reduce the smaller bands.  That solved it almost entirely for a couple of the proteins, though not all.
Also, the DTT should be added before you lyse the cells (even before sonication).

Another alternative is to tag both termini of the protein and do a double purification.  That covers degradation issues and codon issues, though you lose yield.
If those are not the problem, consider a single-terminus tandem affinity purification.

edit:  One other alternative is to test different induction times and temperatures.  Some proteins are expressed well in a stable form in only 3 hours at 37 after induction, but some others do better at room temp for longer periods.

Thank you so much
Yes,I transformed the vector to Rosetta to promote expression, because there seems to be no expression in BL21.
I doubted the possibility of degradation, and added 6-his tag on c-terminal. But i was disappointed after eluting from Ni-NTA, because there was also several bands on SDS-PAGE, then, I had no interest to going on to next step. It's my fault. I will pick up this line again to look for improvement.
Could you tell me the role of NP40, because i have no idea about it
Many thanks!

[Another Jake]

NP40 (Igepal) is just another non-ionic detergent.  If you've got triton already, you don't need it.

Also, when you go to elute from your nickle column, take a series of elution fractions.  I've often seen multiple bands disappear in the later fractions.  Also some of the bands might very well disappear after the subsequent GST protocol.
Good luck.

Edited by Another Jake, 07 May 2010 - 06:55 AM.

[chj]

Another Jake, on May 7 2010, 10:54 PM, said:

NP40 (Igepal) is just another non-ionic detergent.  If you've got triton already, you don't need it.

Also, when you go to elute from your nickle column, take a series of elution fractions.  I've often seen multiple bands disappear in the later fractions.  Also some of the bands might very well disappear after the subsequent GST protocol.
Good luck.

Thanks a lot!