His tag protein purification problem - (Jun/12/2004 )

HI!
You mention:

"As a final option, i scanned for different NaCl concentrations at which the Protein can show binding to Ni NTA.

The Ni NTA to His interaction is so specific that it remains at such high concentrations of NaCl like 1M or even 2M.

The protein that initially creared problem in not binding to any column, bound very well to Ni NTA itself at 1M NaCl in buffer.

I suggest sincerely, if someone has problem in Ni NTA (His tag binding problem) to try higher salt concentrations before concluding any thing."

This can imply that your protein may have hydrophobic properties. I also want to ask if you have a DNA or RNA binding protein? With this high pI it would indicate so! If this is the case, try binding your protein to a heparin column with buffer pH of around 7.0. Hope this helps!

Cindy

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I have had domains that are almost identical in sequence, with the same "theoretical" pI, but which bind on different ion exchange beads. Are you sure the actual pI is 10??? Don't forget there can be a world of difference between theoretical and actual.
Have you tried bind/release trials? get your protein, dilute in a range of buffers from, say, 5 to 9. Add this to ion exchange beads equilibrated at the same pHs (test with both anion and cation beads- yes, it sounds a bit screwy, but you may have patches of charge that makes your protein behave opposite to intuition or theory!). Incubate @ RT for 10 minutes, then spin out the beads. Run S/N as well as beads on SDS-PAGE. That way, you'll have a quick test for a binding pH, as well as an exchange resin. Next step is to use a pH where all of your protein binds (my suggestion is to pick one where all of the protein binds, but don't go too far- you do still want to elute the protein). Prepare some protein at the good pH, add the beads. After incubating as before, split the slurry into several tubes, and add different concs of NaCl to each. Spin out the beads, and run a gel as before. This gives you an idea of the elution conditions.
The whole thing whould take about 1/2 a day. Then you can scale-up onto a column directly.

To take a completely different tack, have you looked at hydrophobic interaction chromatography?

PS.
I have just finished reading all of the posts. It might be a good idea for everyone who is having a problem to direct it to QIAGEN. I'm not saying this to suppress the discussion, because discussion is really, really, REALLY important. It just strikes me that if so many people are having problems with their proteins, then maybe QIAGEN should look at its protocols again! They made the kit, they should be best placed to assist. And when/if you do get a response from them, please tell the rest of us!!! But remeber this, if you're a student, you have plenty of other things to be doing than trying to troubleshoot one particular technique. If it doesn't work (after trying a few options), cut your losses and consider trying something else - ion exchange, hydrophobic interaction, gel filtration, even reverse phase (if you're gentle).

His Tagging should be a really good technique (and it often is), but there seem to be a few cases where it falls down. My protein did, and the only reason I didn't spend a lot of time trying to fix it is because someone else came along and published the structure of the protein.

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The buffers for Ni_NTA column should be checked for their pH everytime before running the column. The wash buffer in particular does not hold its pH very well. Also if your lysis buffer is not at right pH the protein will come out in flow through. I hope you are using the column, samples and buffers at room temperature. Hopw this helps.

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Hi all!
I am working with one 15 kDa calcium binding animal protein having his tag , expressing this in bacteria with pQE-30 expression vector . my protein is coming in insoluble form / inclusion body. i tried to purify this under denatured form with 8 M urea. but it is not eluted even with 500mM imidazole. i used Ni -NTA resin. can anybody suggest me that how can purify my protin and how can i get my protein in soluble form , i tried with different temperature as well as different conc. of IPTG but result was same. if i will change my expression vector i.e. if i use pET vector then will it change the solubility of protein as his tag will be at C-terminal in this case? can anybody suggest me what to do?
with regards :

Ratna Chaturvedi
Ph.D. student

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Hi:
1. The fusion protein can not be eluted at 500mM imidazole maybe due to the quantity of Ni-NTA resin, you may try other company product.

2. Induction parameters:
lower temperature, even 15 degree;
Lower concentration of IPTG;

3.vector:
pET32a (Trx fusion which can increase the soluble level)
sumo vector

GOOD LUCK!

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Hi there,

Well, I guess this is the right place to ask my question...

I have produced a 16kDa C terminally His-tagged protein using a cell free wheat germ system from Roche (confirmed by western). Tried to purify it using Pierce PullDown PolyHis and Amersham Histrap kits without any success. I could only detect low amounts of my prot in the flow through or wash 1. I thought it was not binding to the column but now, after reading a few of your comments, I wonder if my protein is not still stuck to the column... Is there a way to check it?

I used a gradient up to 500mM Imidazole for the elution. Is that enough?

My peptide being quite small, is it really likely that the tag is not available under native conditions?

Thanks in advance

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Hi,

Looks like the His-tag domain of your protein is not expose (hidden) in the expressed form of protein. Denaturing it may help, as you have observed. But you will have to deal with renaturing issue.

CM Sep is cation exchanger. It is negatively charge. Therefore binds to positively charge protein. At pH 8, 8.8, 6.4, is your protein positively charge? Otherwise, try to use lower pH or switch to a anion exchanger like DEAE or Q Sep. It is helpful to scout for the right media at different pH using loose media in test tube.

Henry

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Hello, Im working with recombinant proteins and I would like to know if DTT will interefere with the bounding properties of my Niquel-resin.

Thanks a lot

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You might want to do following things

1) make sure there is little or no imidazole in your binding buffer ( I usually keep it to 2mM)
2) c terminal taggs are more difficult , you might want to try pet30 vectors from novagen which have his tagg at both ends
3) instead of column try batch purification , to your lysate add 3ml Ni nta bead / 500 ml culture and incubate in cold room for 14 to 18 hrs . next day spin at 2000 rpm to reomve super and wash with wash buffer 2 -3 times , elute similarly ( collect every wash and run gels)

hope that helps

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Hi,

I actually tried with no imidazole with no difference in the results. I think I will give the batch purification a go. BTW, why do you say that C terminal tags are more difficult?

Thank you for your answer!

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