I'm currently doing protein purification. my protein size is around 42kDa, however, my protein is attached with fusion tag which are Trx-tag and His-tag at the N terminal. I did optimize my buffer condition to cleave the his-tag. I also did try to incubate at 5C, 20C and room temperature but nothing works on my protein. The thrombin only succeeds in cutting half of my total protein. I suspected the difficulty in cleaving the thrombin site is due to the hydrophobic region from Trx-tag covering the cleavage site. any suggestion is welcomed.
I am not sure if you have used any reducing agents or not, since it isn't mentioned, check the buffer condition. If the hydrophobic region has predominantly cysteines try to use reducing agents like BME or TCEP. Similarly, for other predominant amino acids use detergents. Using such agents will allow exposure to the cleavage site. Good luck.
You could:
a) be satisfied with the 50% cleaved protein that you can recover in the flow-through after passing the cleavege mixture on a Ni-NTA column. Just process more starting material (cell pellet) if needed.
b) try to improve the yield of cleavage by adding more thrombin or increasing the cleavage time
Are you sure that the problem comes from the Trx tag ? It may also be that your target protein is hiding the processing site. This is a frequently encoyuntered problem. The amount of processing enzyme/time of reaction varies greatly with different target proteins
Dear Siti
did you tried to load the protein in a SEC coloumn to check the aggregation state? Because in my experience, incomplete tag cleavage is often due to protein aggregation which make the digestion sites buried.
In this case i suspect that you have to:
- try different buffers (eg change pH if is close to your pI)
- Try to change the expression conditions (eg reduce the induction temperature) to see if you may obtain less aggregated protein.
good luck
Manuele
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