hello every one, i am expressing lipase protein with N.terminal His tag. some have His tag is not working. i am loosing great amount of protein when i do nickel affinity cinematography, i changed my His tag to C-terminal, my protein lost the activity. so i continued to work with Protein contain N-terminal his tag. but when i purify my protein further, lot of junk protein is still remained in the sample. can any one suggest me the solution for solving this issue.
What vector you are using? I have used pET28 vector with N-terminal His tag. Proper washing is required prior to elution with low concentration imidazole wash buffer(10-25mM) to get rid of unwanted protein bands. Elute your protein with 200-300mM imidazole concentration.
Hope it will work.
thank you for the reply.
i am using pET 28A vector. induced with with 1mM IPTG. i am equilibrating with 5mM, washing with 50mM and eluting with 400mM imidazole. at this concerntration of washing i am getting 50% junk protein in elution.
Dear Dachuri,
You can assay with an Imidazole gradient (from 20mM to 500mM). You probably will lose some of your protein in the first elutions, but the last ones should be cleaner.
Good luck
hello Cristián Quintero.
i have tried the gradient separation. its not working. the great problem is protein most of the recombinant protein is not binding to the column. protein recovery is poor.
Hi Dachuri,
Other option is to change the tag, you may consider GST- or Flag-tagged protein. For GST is easier to purify and release the GST after the purification.
Best regards
hello Cristián Quintero,
for the first time we used GST. but protein is secreting as inclusive body. after recovery, it lost the activity. As this protein is having many extreme characteristics, i want to study mechanism in detail, so i need mere concentrated and very pure protein. is there any other way by which i can express this protein in high concentration?
we tried to concentrate the protein, some have it worked. but now we made mutation the level of expression gone down. its hard to recover the protein.
Hi Dachury.
Did you try changing the host? I know is a lot of extra-work, but insect cells are a good option. I don’t know if your need or not some post-traductional modifications.
With the mutated version of the protein, I guess you assayed other culture conditions also, to increase the amount (Temperature, induction time, inductor concentration, etc).
hello Cristián Quintero.
my protein is a prokaryotic protein, i am using BL21. i have assayed all the conditions you mentioned.
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