Protease inhibitors

bhjelle at carina.unm.edu bhjelle at carina.unm.edu
Wed Oct 14 08:36:38 EST 1992


In article <9210131601.AA11710 at net.bio.net> NANTEL at UNCVX1.BITNET writes:
>
>I'm fishing for information concerning the use of protease inhibitors during
>the purification of MBP-fusion proteins from E.coli. PMSF doesn't seem to
>have any effects on the quality of my protein preps. Furthermore, when I
>use antipain + leupeptin the efficiency of the purification step through
>the amylose column is greatly reduced (7-10 fold lower yields). I feel
>that protease inhibitors are necessary since one of my fusion protein is
>chewed up from 95 to 80 kD during the purification.
>
>I am faily new to the world of protein purification (yes, another molecular
>biologist doing biochemistry without a licence) and any suggestions would
>be most welcome.
>
I could have written this same message. Only the MWs would be
changed to protect the innocent. I'm using trp E fusions and
getting rather severe proteolysis problems. One "solution"
I was considering was to go to pMAL (MBP fusions), but now I'm
not so sure :-).

I am working now on scaring up some protease-deficient E. colis
(lon-, hgpr-) but am told these are pretty tough to work with
(frequent reversions). Will post if this seems to help with my
constructs.

You didn't mention whether it is possible that your fusion is
getting chewed up in the cell, as opposed to during extraction.
I'm pretty sure my problem is intracellular, since I can lyse
the bugs right away, immediately boil in SDS, and see the exact
same degree of proteolysis.

Now I wish I had spent as much time worrying about protein
chemistry as nucleic acids...

Brian



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