Thrombin - pDEST20/GST-Baculovirus - Cloning and Protein expression (Aug/17/2006 )
Does anyone work with pDEST20/GST vector for expression of GST-fusion protein in Baculovirus/Insect cells.
I got this from Invitrogen and there is doubt if thrombin cleavage site is present or not?
I checked the sequence and its a yes or no type answer because the aa seq. is of the variable kind.
Any insight into this will be greatly appreciated
I don't know about the plasmid in question, but I was thinking that you could just design the forward primer with a thrombin site in it. That way, if you ever decide to move your insert to a different system, you can take you protease site with you...
you may ask invitrogen and give them the batch number of your plasmid
or design a trhmobin cleavage site as mentionned before.
or sequence the interesting part of the plasmid (which will give you the answer for now and the future).
The reason for asking is that I am already down the pipe line for my protein expression and the his-tag version I tried before does have a cleavage site. I thought that the GST version should too. But now, I am following the rule governing the thrombin cleavage site and am in the process of checking it out with the enzyme; (A-B-Arg//-X-Y). I was just hoping to see if anybody had specifically tried pDEST-20 before.
Designing is not a problem, just the time issue.