Can i know the estimated protein size of your targeted protein without tag?
I am a little curious, had you try this before?
1) Load recombinant protein into the Ni-NTA or MBP column
2) Wash the unbound flow through
3) Add in your HRV3c (around < 2 CV) into your column. Hold the system for 30 minutes (or any amount of time desired for the protease to do it's work)
4) Instead of using elution buffer, perform an isocratic elution (for around 5 CV) using column / running buffer and start collecting all the fractions
5) Pull all your fractions together, perform size exclusion chromatography (Superdex Peptide PC 3.2/30 -or- HiLoad 16/600 Superdex 75 pg) to separate your HRV3C and your protein of interest.
CV : Column Volume
Thank you very much. My target protein without tag is about 28kD.
1) I used Ni-NTA and MBP column to get my quite pure protein with tag. Then used HRV3C(it has his8 tag) to cut the tag. After this, I load the mixture onto Ni-NTA again. Theoretically as long as the NI-NTA medium is enough, the uncut protein, the tag and the HIS-HRV3C will be attached, and my protein flow through. But there was a part of tag also flowed through. So I planned to do the on-column cleavage use these two kinds of column. 2)After loading my protein lysates onto the columns and washing the unbound contaminant, I added HRV3C into the columns and hold them for about 12h. For Ni column, nothing flowed through. Maybe because the number of His. But for MBP column, a small part of tag also flowed through although most can bind. 3)Also, I tried resource Q column to separate the tag and my protein. The result was confusing. Although I got two peaks, each peak contains the tag and my protein,and it seems to be the same ratio. Maybe I didn't get the appropriate condition for this.4)I also tried gel filtration(Superdex 75 or Superdex 200). All of the proteins are in the high aggregation peak, include the MBP tag.......I am sorry about that I do not know how to attach the result image.... I am very frustrated.
Thank you again for your warm-hearted reply.
Pardon me, initially I thought your are purifying a small protein. Just try my best to help to troubleshoot....do you do complete desalting before you reload your protein back to Ni-NTA/ Resource Q / MBP column?
IMHO, I would stick to your first approach. I would suggest to load total protein which is 50-70% of the total protein binding capacity of the column. You can attach 2x column together to increase the total binding capacity, or purchase a bigger column, to avoid the escape of tagged protein.
Since your second approach only got a little of tag in the flow through (not tagged protein), I believe with another round of Superdex 75, you can separate both away.
For your third approach, I can't tell much without knowing the chromatogram and the buffer pH you are using.
According to :http://www.sinobiolo...ase-g-1646.html it says that the Protease is around 22kDA (and I believe your protease might be the almost the same size), while your untagged protein is 28kDA. Even with Superdex 75, although you can reduce your protein load or dilute your protein concentration, it will still be quite difficult to separate both protein with such close sizes.
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