Hello everyone,
for invitro cleavage of recombinant protein, i need to use Enterokinase. Protein is getting cleaved properly, but i think its being degraded as well. Anyone ever used Protease Inhibitor which is compatible to Enterokinase and don't hamper its activity?
please let me know
Thanks in Advance
As enterokinase is a serine protease, avoiding inhibitors that target such enzymes should be avoided. Reagents alkylating thiols such as iodoacetamide will inhibit SH proteases will probably notaffect enterokinase, but they may react with your protein. The safest would be chelators to block metalloproteases. Otherwise, you may try to process the prep rapidly and in the cold, of course.
Hi Aditya, AFAIK corn trypsin inhibitor (CTI) is not a good inhibitor of enterokinase (horrible nomenclature!) but inhibits many serine proteinases.
Dear Aditya
Did you tried to incubate your protein in the same buffer but with out enterokinase to check in the degradation is due to other proteases or to an aspecific digestion of enetokinase ?? To my knowledge enterokinase some time has problem of specificity https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2441903/
Generally i prefer tev for removal of n terminal tag and fatt.xa for removal of c terminal tag.
Good luck
Manuele
Thanks to all of you. I have got some ideas and i will play around them.
I need also to use EK to cut mys tag, Novagen recommend digest at room temperature several hours, have anyone tried to diget at 4ºC? or any other suggestion to better preserve the recombinant protein?
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