GST fusion proteins and dnaK

Bipin K Dalmia bipin at iastate.edu
Mon Jun 27 22:05:09 EST 1994


i have a 70 kDa protein (probably dnaK, according to the GST manual)
being co-purified with my fusion proteins (two of them). it stays
associated with my protein and not the GST partner during the
purification, dialysis and repurification on the glutathione-sepharose
column. the uncleaved fusion protein and the GST portion of the cleaved
fusion protein bind again to the GSH column leaving me with two bands at
about 50/50 ratio (and also the thrombin used for cleavage). when i run
this repurified fraction over a mono-q column, eureka!! the 70 kDa
protein binds while my protein flows right thru. 

why would the association between my protein and the 70 kDa protein
remain intact thruout except on the mono-q??

also, any ideas on how to get rid of the thrombin?? what is the pI of
thrombin? 

one last thing: i have lots of degradation?? any particular e. coli
strains that you've found useful in that respect?

bip
-- 
bipin k. dalmia               the other night i was lying on my bed, looking
bipin at iastate.edu             up at the beautiful stars, and i said to myself, 
n2.bkd at isumvs.iastate.edu     'where the F*CK is my ROOF !!'
--



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