His-tag protein purification

Ian Goodfellow plxigpg at pln1.life.nottingham.ac.uk
Tue Apr 30 10:57:41 EST 1996


Dear all,

I have another problem that needs sorted out:

I have been using Qiagens pQE system for the overexpression of a His 
tagged protein. Basically I have PCRed  a 1.2Kb fragment and transformed 
XLI blue and SG(??)pREP4 (the strain they provide). I isolated 3 
independant isolates 2 in SG and 1 in Xli - all were cloned into pQE32. 
When I induced the cells I expected a 40KDa (ish) protein. However what 
I did see was a 60KDa and a 40KDa. I then purified them under denatirung 
conditions (6M urea) and both purified.
 What could the 60KDa protein be? Do you get any read-thru the 
terminators?? I think this is unlikely. The PCR fragments are of the 
correct size.

Thanks in advance


Ian
PS could you please email me direct as I have problems getting access to 
the newsgroup
 





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