I totally agree with Patric.

I'm currently working on a his-tagged protein purification from Sf9 cells. There was lots of precipitate after every incubation step (incubation on agarose, wash steps...). But in the end, the protein was in the elution fraction!

I found some information in "The QIAexpressionist"-Manual from Qiagen:

"...Expression of 6xHis-tagged proteins secreted directly into the medium can pose some problems. Media used to culture insect cells usually have an acidic pH (6.0–6.5) or contain electron-donating groups that can prevent binding of the 6xHis-tagged protein to Ni-NTA...."

But the more interesting part:

"...Dialysis of the medium against a buffer with the appropriate composition and pH (8.0) similar to the lysis buffer recommended for purification under native conditions usually restores optimal binding conditions. Note that depending on the media used a white precipitate (probably made up of insoluble salts) can occur, but normally the 6xHis-tagged protein remains in solution."

Nevertheless, I'm afraid that I lose protein with any precipitation...I'm thinking of a pre-incubation or dialysis step before the purification to get rid of These precipitates.

You could try to dialyse your sample against a suitable buffer. I you still find precipitation, find out if your protein is in the precipitate of in solution. Maybe this precipitation is already your first purification step ;-)

I totally agree with Patric.

I'm currently working on a his-tagged protein purification from Sf9 cells. There was lots of precipitate after every incubation step (incubation on agarose, wash steps...). But in the end, the protein was in the elution fraction!

I found some information in "The QIAexpressionist"-Manual from Qiagen:

"...Expression of 6xHis-tagged proteins secreted directly into the medium can pose some problems. Media used to culture insect cells usually have an acidic pH (6.0–6.5) or contain electron-donating groups that can prevent binding of the 6xHis-tagged protein to Ni-NTA...."

But the more interesting part:

"...Dialysis of the medium against a buffer with the appropriate composition and pH (8.0) similar to the lysis buffer recommended for purification under native conditions usually restores optimal binding conditions. Note that depending on the media used a white precipitate (probably made up of insoluble salts) can occur, but normally the 6xHis-tagged protein remains in solution."

Nevertheless, I'm afraid that I lose protein with any precipitation...I'm thinking of a pre-incubation or dialysis step before the purification to get rid of These precipitates.

The others bring up some good points. I’ve also observed that there can be components in certain insect-cell media that inhibit good His-tag binding to Ni chelating resin. As suggested, buffer exchange before the Ni column can help. An alternative to dialysis is a quick ion-exchange column with a wash and then a step-elution before your affinity column. You just need to determine which type of IEX resin your protein binds to.

Hi Patric

I am afraid that I can't dialyse the medium if I express the protein in a large scale like several liter of the medium. I wonder if I should add some imidazole (eg 20mM) in the medium in order to rise the pH. Have you tried this?

Hello Charlotte

I have clone the gene into the pAc67A vector which carries a N-terminal signaling peptide for the secretion, and the His-tag is located in the C-terminus of the protein. The signaling peptide should not affect the his-tag.

Dear Mattew,

Ion-exchange column could be a good idea. But I am afraid that my interested protein may not bind well as lots of other secreted proteins exist.

peptide could be removed by SF9 cells during the secretion.

Dear Peter,

in your case of several liters of medium I would prefer the ion-exchange chromatography suggested by Mattew. This will concentrate your protein, you will get a first purification and maybe rid of the stuff that precipitates. Afterwards you can and will have to dialyze your sample against a suitable buffer for Ni-NTA chromatography.

Kindly do the following

clarify the culture media by centrifugation at 4000 rpm

further clarify by 0.2 micron filtration

concentrate by Tangential Flow Filtration {tff} using Millipore spiral wound cartridges

dialfilter using your buffer of choice 10X with tff cartridge.

fun this on NiNTA beads..will work absolutely fine...

we do 10L to 50L scale with SF900II media / SFX insect media and works great

Adding imidazole in your media may reduce the binding of His tag to Ni-NTA further.

The precipitate could also be due to your protein if you are at it's PI. So I would raise the pH higher, to try to stay away from its PI if that is the cause. We typically did not have this problem, ie of not sticking to Ni NTA, with collected media from SF9 cells. So maybe you can check to see if your protein is the precipitate first. If it is not, then raise the pH further. If it still doesn't stick, then see the research post as Charlotte has suggested. It may just not stick. I don't know if ion exchange would help if it is precipitating at the pH you attach the protein to the column. If it is a solubility issue at pH 7.4, then you may need to find the proper buffer. Sometimes even switching from PBS to Tris helps.

For changing the buffer, once you are at pH 7.4 or 8, all the histidines are neutral on the protein, so in order to change total charge of the protein, you may have to go to pH 9 or above. If you add a small amount of imidazlole, this will raise your pH. Your protein should stick under these conditions unless the His tag is unavailable for binding, ie it is not accessible because of the way the protein is folded. If this is the case, then you can try adding 6M urea, and see if it sticks under those conditions. If it does, you will need to refold, or change the construct by altering the spacer on the His tag if you can't change it's position.

Thank Muralidhar Reddivari for your suggestion. Unfortunately, our department does have this devise for the dialysis. That is why I have to seek for other ways to address the problem.

Hi Marcia

The pI of the protein was estimated by Expasy, which is around 6.4 so I don't think it is the pI problem. Someone suggested that SF900-III medium may contain histidine-bleaching IMAC reagent. This results in the leakage of Ni ions. So I would like to know which medium you use for the expression of secreted proteins for insect cells.

I would adjust the pH slowly with stirring, and see if the ppt is your protein. If it isn't, then I would centrifuge, and load your material onto the column to do the purification. I don't know of other medias for SF9 cells, but I can ask if you want and get back to you, or you can contact Robert Petrovich or Gary Powell on ResearchGate.

the pH of insect media after 3 days of culture is about 6.3. the pI value is an estimate based on the assumption that your protein AA chain is linear. use a TFF module with appropriate MWCO to change from culture media to a suitable buffer, then try IMAC. it will work. We have always done that for secreted proteins [about 100 + proteins] and it works wonders. that is a very simple solution. also make a construct with the tag position changed and try that, that might work better.