You can try Genescript. They do commercial protein expression and purification.

Cant u try any other tags???

GE site says your column has a dynamic binding capacity of Approx. 6 mg Strep II-tagged protein/ml medium when they tested with GAPDH-Strep(II), Mr 37 400. Even if you get 50% of that you still could good amount needed for your studies in 2-3 purification.

But, if your yield is too low then you should optimize the expression. You could look up some excellent suggestions made in a parallel question @ https://www.researchgate.net/post/How_do_I_know_my_His-tagged_protein_is_binding_to_the_Ni-NTA_column_or_not

You could additional ion exchange and gel-filtration step to remove contamination.

I dont have first had experience with Strep tag, but some times adding second tag makes life easier. Eg; Adding a his tag at c-terminal to MBP-tagged protein made purification lot easier for me.

I have suggested these thinking you are expressing protein E coli.

Good luck

Thanks so much for the suggestions guys; I use leaf infiltration; I have optimized expression; it is under constitutive promoter and highest expression is achieved after 6days post transfection; my proteins have both Strep and His tag; but my yield is so low after first round of purification - it is hard to think about a second round :(

If you have high expression but low yield after only one chromatography than is something wrong with the chromatography or between. Do you use protease inhibitors? Did you check activity in flow-through to be sure your protein is binding to the column?

Are you studying a cytoplasmic protein or a membrane protein? Do you recognize any tissue specificity, i.e. is there higher expression in the mesophyll or epidermal cells? Do you have antibody for this protein?

Do you know that your protein is being expressed? Have you performed a western or tissue blot to obtain information about yield and location prior to performing the chromatography?

If you have both His and strep tags, it might be better to try His tag first, strep tag usually has lower efficiency.

Before you try yet another strategy, it would be very valuable to determine the efficiency of the leaf infiltration. You state that you have optimized the expression. What data can you present to that effect relative to expression in the leaf.

If you just want protein, you do not need to perform the leaf infiltration.

If you want protein, you should consider placing your construct under the control of an inducible promoter.

Again without a little more detail it is not clear what advice should be provided.

I agree with Fedora. You should try an inducible promoter. Furthermore, have you compared protein in cell free extract to semi purified samples? I ask because could there be an issue with cell lysis. Otherwise, I agree with other about the histag, purifcation is simpler and can be easier to modify to remove protein impurities.

The question is- are you inefficiently producing the protein, or inefficiently purifying it?

Strep tags can give very high levels of purity, but in our hands the capture may not also be very efficient. Also, in your post you say that you have both strep and His tags- are these on the same end of the protein, or is the strep tag internal to the His? Internal strep tags are bound with lower efficiency than terminal tags. Do you have protein in the flow through from the column (ie, is the column saturated)? This can easily be detected by using a Strep-Tactin conjugate to detect the Strep-tag (for instance, the conjugate sold by iba life sciences).

If you have a His tag, I would try using that for the initial capture, and then follow up by purifying via the Strep-tag if necessary.

Can you clarify what is the source of protein. Is it expression in shake flask or from expressed plant leaves or tissue?

Oh God, at least read the whole discussion! Rowshon wrote her expression in leafs is optimized giving high activity.

Rowshon Begam · 6.09 · University of Alberta

Thanks so much for the suggestions guys; I use leaf infiltration; I have optimized expression; it is under constitutive promoter and highest expression is achieved after 6days post transfection; my proteins have both Strep and His tag; but my yield is so low after first round of purification - it is hard to think about a second round :(

We normally use plant as platform for producing recombinant proteins. We are a small company from Italy if you are interested we can manage a feasbility study for the purification of your recombinant protein. Are you working with tobacco or benthamiana plants? What are the yields of your recombinant protein in leaf fresh weight?

Hi Linda, thanks for the note; I used N. benthamiana; I can not give you any quantitative number for yield right now; I tested 3, 4, 5, 6, 7 days post transfection; in the western I find most intense band on 6th day; so I continued harvesting tissues 6days post transfection; could you please send me an e-mail to [email protected] with the detail of how we can arrange an agreement with you for the feasibility study; I will talk to my PI; and we can go from there.

@ all: thanks so much for your thoughtful response; I learn something new from each of your comment; thanks again.