Se-met protein question

Marjolein Thunnissen marjo at milvus.molbio.su.se
Thu Jun 26 09:22:01 EST 1997


Dear all,

We are currently trying to purify a Se-met protein (85 kDa.) but we are
having unexpected problems. The overexpression  of the protein went ok
but now in the first step of the purification the protein get attached
to the column and can only be removed using harsh methods. The column we
are using is a butyl-sepharose column (hydrophobic) from Pharmacia on an
FPLC system. The normal eluation is by using a ammonium-sulphate
gradient from 0.5M to 0.0M. The protein always bound tightly and came
off at the end of the gradient. The Se-Met variant can only be removed
by using 0.5M sodium hydroxide, indicating precipitation on the column. 

In the buffers we are using (phosphate pH 7) we have 5 mM DTT and 1 mM
EDTA present. We have tried using Tris pH 8 but there is apparently
still some precipitation on the column.

Has anyone had similar problems with a Se-Met protein before? We were
warned before hand that the solubility of the protein might be affected
by the Se-Met substitution and that retention times on columns might be
changed, but not of something like this.  

Tnank you
Marjolein

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Marjolein M.G.M. Thunnissen            phone:  +(46) 8 674 7019 
                                       fax:    +(46) 8 152350
Lab. of Molecular Biology,             email:  marjo at milvus.molbio.su.se
Arrhenius Laboratories F3,             
Stockholm University
S-106 91 Stockholm
Sweden

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