Nightmare with GST fusion protein

Bernard Heymann bheymann at bragg.bio.purdue.edu
Sat Nov 19 15:56:07 EST 1994


In article <Pine.3.89.9411182228.A2259-0100000 at eukaryota>,
massonjy at rsvs.ulaval.ca (Jean-Yves Masson) wrote:

> - Different hosts (XL-1 blue, DH5 alpha, HB101, BL21 and AD202)
> - Time course: my protein is cleaved in less than 30 min after IPTG 
> induction (then my fusion protein is 31 kD instead of 66 kD)

Do you see an uncleaved fusion protein before or just shortly after
induction?

> - Different protease inhibitors
> - Growth at 30 C instead of 37 C.

This seems to be a general problem that crops up lately. In my case I also
see a 28 kD product that must be the GST part, since it has activity, as
well as larger products. Through various experiments I've become convinced
that the multiple products are NOT due to degradation, but rather poor
readthrough of the transcript. I did a few experiments to change the mRNA
secondary structure at the linker region, obtaining significantly different
products, although not much of the full-length construct. So at the moment
I'm also stuck.

-- 
Bernard Heymann
bheymann at bragg.bio.purdue.edu



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