Yeast protease inhibitors

Thu May 4 16:59:34 EST 1995

	I'm looking for help in creating an effective protease inhibitor
cocktail for yeast extract - I am expressing a small soluble enzyme in
Saccharomyces with a C-terminal affinity tag.  I get plenty of expression,
but at each step in the purification I see significant amounts of active
enzyme which has lost the tag, as confirmed by SDS-PAGE.  The inhibitors I
am using in mybreaking buffer (for BeadBeater disruption) are:
		5 mM phenanthroline
		1mM pefablock
		10 microgram/ml pepstatin A
		10 microgram/ml leupeptin
		1 microgram/ml aprotinin

	I have also considered adding EDTA in the breaking buffer but must 
remove it before an Ni-NTA binding step.  If anyone has any experience in
optimal inhibitor mixtures and concentrations for yeast whole cell extract
(or references for the same), please let me know.
	Also, has anyone ever heard of the effects of various C-terminal
amino acids on protease susceptibility (C-end rule?)?
EARN at					Division of Biology
Abelson Lab						Caltech

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