Hi all, I want to obtain a protein of interest without the first methionine and I would like to know if there is a expression protocol to optimize the methionine aminopeptidase of E. Coli or it is possible to eliminate this methionine after the purification.
And another question: How to remove the N-term His-tag and recovery of the intact protein of interest with no extra residues? Because TEV and 3C protease leave extra residues.
Thank You.
Hi there,
Methionine aminopeptidase will do the job for met removal:
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/4/m6435dat.pdf
If you absolutely don't want to get any extra aa after cleavage of the Nter 6his you'd better use enterokinase or factor Xa which cut exactly after their specific recognition sequence and read the page 10 of the pET manual which is describing some cloning strategies to do so with pET41, 42, 43 or 44 derivatives. You have access to the manual with the following address:
http://richsingiser.com/4402/Novagen%20pET%20system%20manual.pdf
all the above answers are very good. For another alternative and one that will cleave efficiently and leave no extra amino acids at the N-terminus, you could look into SUMO containing vectors. These have a His tag followed by the SUMO protein. It will enhance expression and folding of your cloned protein which can then be released using SUMO protease.
A very efficient system is the Expresso one from Lucigen.
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