protein crystals?

Soenke Behrends behrends at plexus.uke.uni-hamburg.de
Tue Aug 31 11:17:41 EST 1999



Dear netters,

we have done overexpression of a cytosolic 
hemoprotein in E. coli, got nice expression and
have done DEAE anion exchange chromatography
of the ultracentrifuge supernatant as first 
purification step. During the elution with 
a buffer containing 50mM Hepes pH 7.4, NaCl 
450 mM, DTT 5mM, Pefabloc 1 mM (Merck, PMSF 
like stuff) we have noticed brownish stuff at
the tip of the elution tube. We have taken 
some stuff from the tip onto a glass slid 
and under the microscope 
there were apparent big NaCl crystals 
and much smaller yellow - brownish crystals.  

The fractions containin our protein looked nice 
but there were still some contaminating proteins.
I would say maybe 70 % purity. Protein conc. was
2.5 mg / ml. 

I know that you normally need much higher protein
purity and concentration to get protein crystals. 
Does DTT form brown crystals or could it be nucleic
acids or something else non-protein like from the 
coli? 
On the other hand could the brown yellowish color 
be compatible with a hemoprotein? And maybe the 
protein under investigation just crystallizes like 
hell?

I would be very happy about any advice or comment
or guess or hint on literature. 

Thanks a lot for your time and help
Soenke 








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