Re: [ccp4bb]: soluble aggregation of proteins

["Francisco J. Enguita" <fenguita@itqb.unl.pt>]

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Dear Janet :

You give us only a little bit information to discuss your problem ..... 

As far I have understood you get aggregation problems of the proteins after
Xa factor digestion of the fusion protein (is it true?). If this is your
problem, try to increase ionic strenght in the resuspension buffer (NEB
manual for the pMAL systems says 200 mM NaCl, so try 300 mM). You can
include glycerol at 5-15 % in the column buffer as well to avoid aggregation.

Does your protein have disulfide bridges ?. If this is the case, I suppose
you can include DTT or beta-mercaptoethanol in the buffer to avoid
intermolecular disulfide bridge formation.

I hope it helps. Good luck.

Francisco.



>Dear all,
>
>We are expressing proteins here in E. Coli in the pMALp2 vector as
>maltose-binding fusion proteins. This is a periplasmic expression and we
>have found that our three proteins in the 22-38 KDa range all aggregate
>in solution. 
>
>Any suggestions from your experience to treat this type of problem?
>
>Thanks,
>
>Janet.
>
>-- 
>Janet Moloney, Division of Structural Biology,
>Wellcome Trust Centre for Human Genetics,
>University of Oxford, Roosevelt Drive, 
>Oxford OX3 7BN, U.K.
>
>Tel: 0044(0)1865 287551	   Fax: 0044(0)1865 287547
>
>
=================================================
Francisco J. Enguita, Ph.D.
Protein Crystallography Laboratory
Instituto de Tecnologia Quimica e Biologica (ITQB)
2781 - 901 OEIRAS
PORTUGAL
Phone : (351)-214469662/214469438
Fax : (351)-214411277
E-mail : fenguita@itqb.unl.pt
==================================================