After having trouble expressing a prokaryotic flavoprotein due to inclusion body formation, we resorted to SF9 insect cell expression. We were able to finally obtain full length, soluble protein. However, the full length protein will not elute from the nickel resin. We have ranged the imidazole concentration from 200mM to 1M with no luck.
- We have been using protease inhibitor cocktails and sonication for lysation. The lysis buffer / binding buffer is 20mM Tris, 250mM NaCl, 10mM imidazole, 5% glycerol at pH 7.4.
The theoretical isolectric point of the protein is around 9.2
Could this elution problem be due to protein aggregation in the resin, possibly caused by imidazole? Could the problem lie with the binding/wash buffers? We have tried increasing the salt concentration of the buffer to .5M with no luck.
Any advice would be appreciated, thanks.
Try with different concentrations of imidazole at a lower pH
We got it to elute from the column by putting 2% sarkosyl in the buffer although the protein did crash out of solution later.
hey !!! I work on a flavo protein fuse whit HisTag and MBP. I eluate the protein whit 400mM of Imidazole!!
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression