protein crystals?

Brett William Lennon bwlennon at umich.edu
Tue Aug 31 16:25:26 EST 1999


Yes, you could have protein crystals.  The protein concentration and
purity necessary for protein crystallization is quite dependent on the
protein in question.  I've heard of one protein (the name of which
escapes me right now) that could actually be crystallized by slicing liver
and leaving the slices exposed to air.  But as you state, in many cases
high purity is indeed necessary for crystallization.  A good reference for 
protein crystallization is Preparation and Analysis of Protein Crystals by
Alexander McPherson.

You mention having seen NaCl crystals under the scope.  NaCl is a
precipitant used in macromolecular crystallizations, and at least in the
sample you're observing, it's saturated NaCl created by evaporation of
water from the elution buffer at the tip of the tube.  This same
evaporation could drive up the protein concentration at the tip of 
the tube well above the 2.5 mg/ml you measured in the fractions
themselves, which combined with the high concentration of salt could very
well lead to protein crystallization.  The color of the unknown crystals
is also consistent with a heme protein, and McPherson's book mentions that
metalloproteins as a group tend to crystallize rather well.

It isn't clear from your posting if there is any liquid left in the sample
you observed under the scope.  If not, add some 50 mM HEPES pH 7.4 + 5 mM
DTT saturated with NaCl to the sample to provide some liquid without
(hopefully!) dissolving the brownish crystals.  Use a small pipettor like
a Gilson P-10 to harvest as many of the brownish crystals as you can.  Put
them together in a fresh drop of ~50 microliters or so of the saturated
NaCl solution to help wash away any residual uncrystallized protein as
well as the crystals of NaCl.  Repeat the washing step.  Harvest
the washed crystals, dissolve them in SDS-PAGE sample loading buffer and
run them on a gel to see if they run where expected for your protein.  If
you get enough of these crystals, you could even dissolve them and use
UV-vis spectroscopy to help identify the sample.  If the crystals are a
heme protein, this could be quite useful.  If these crystals turn out to
be your protein, you might want to consider crystallization as a
purification step.

Good luck!

Brett

-------
Brett W. Lennon
Department of Biological Chemistry
The University of Michigan
bwlennon at umich.edu



On August 31, 1999, behrends at plexus.uke.uni-hamburg.de (Soenke Behrends)
wrote:

>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.  

<remainder snipped>



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