GST Problems - an idea

rpgrant at molbiol.ox.ac.uk rpgrant at molbiol.ox.ac.uk
Wed Nov 23 08:38:17 EST 1994


Skimming the recent postings on GST problems, reminded me of the following 
experience which may or may not help some of you.

I was trying to express a 280 kDa fusion in maltose-binding protein, and found I 
had very low levels of fusion protein - not surprising, perhaps :)
Antibody-mapping with abs of known epitope which reacted all along the foreign 
protein and with MBP showed me that I was getting premature termination of 
translation/transcription.  This was resolved by supplementing the media with 
1% glucose and 10 mM Mg sulphate.  The induction temperature was reduced to 
25C and presto!  full-length undegraded protein.  The basic media was standard 
2XTY before supplementation.  I later added 1% glucose routinely to a smaller 
fusion (90 kDa) prep to aid production.
Hope this helps...... ;)

-- 
Richard P. Grant             
University of Oxford, Nuffield Department of Obstetrics and Gynaecology
John Radcliffe Hospital Maternity Department, Headington 
OXFORD OX3 9DU. UNITED KINGDOM.      Fax +44 1 865 69141



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