proteolysis during expression in bacteria

Emir Khatipov ekhatipo at NOSPAMmidway.uchicago.edu
Mon Feb 19 21:14:39 EST 2001


I remember NEB's manual for pMAL system (check out their website) has a
discussion in the troubleshooting section on in vivo proteolysis and
recommends strains to deal with the problem. I guess you can download the
manual in pdf format.
As for your few problematical proteins, are they all prokaryotic? It has
been discussed earlier in this group and at bionet.molbio.methds-reagnts
that in case of heterologous expression you may also have a codon preference
problem that could be solved with the help of BL21s expressing additional
copies of tRNAs that are rare in E.coli (RIL and RP strains - from one
letter codes for the problematical amino acids) . Again, you can find info
at NEB site (no affiliation).

Hope this helps.
Emir


"Suzanne Elsasser" <selsasser at mediaone.net> wrote in message
news:3A8F3D3A.9D4C5D14 at mediaone.net...
> Greetings All,
>
> Currently, I am expressing a number of GST fusion proteins
> and am experiencing proteolysis problems.  Upon
> purification, I obtain mostly truncated forms of a few of my
> fusion proteins. (The others look great.)  These proteins
> are being expressed in BL21(DE3) cells, the vector backbone
> is pGEX-2TK, expression is induced at 25 degrees, and the
> extracts are prepared in the presence of protease inhibitors
> (aprotinin, pepstatin, AEBSF and EDTA) by French press
> lysis.  I suspect that the lysis occurs in vivo, and am
> curious about whether there are bacterial expression strains
> engineered to contain fewer proteases than BL21(DE3) cells.
> I would be happy to hear from anyone who has successfully
> troubleshot this particular problem.
>
> Thanks a bunch,
> Suzanne
> _____________________________________________________________
>
> Suzanne Elsasser
> Home: 617-354-2069, selsasser at mediaone.net
> Lab: 617-432-1519, selsasser at hms.harvard.edu
>
> "There are four things that we want from ourselves and the
> people in our lives:  passion, compassion, humor, and
> style."
>
>







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