His tags & best second codons

James Bassuk bassuk at u.washington.edu
Fri Sep 15 02:09:34 EST 1995


I put my His-tags at the C-terminus to avoid any complications at the
N-terminus.

In addition, the His-tag at the end allows purification of full-length
proteins.


********************************************************

   Jim Bassuk, Ph.D.
   Dept. Biological Structure, Univ. of Washington
   NetworkAddress:  Bassuk at U.Washington.Edu  

   'Did I say what I meant or did I mean what I said?'

********************************************************



On 11 Sep 1995, Christopher Walentas (Bioc) wrote:

> Hi.
> 
> I've been trying to express a tagged version of glutathione reductase
> but to no avail.  The wt expresses very well (100mg/L) from a Ptac
> (pKK223-3) vector (the tag isn't for straightforward purification
> purposes, but has a "higher" task).  At first I tried MetHis6...
> but that didn't work (no protein could be seen on a gel, or by
> activity).  I was then going to try sticking a few more neutral
> residues between the Met and the tag itself.  pET seems to fancy
> MetGlySerSerHis6, while QIAGEN likes MetArgGlySerHis6.  Does anyone
> have any personal favorites, or any comments upon the realtive
> levels of expression between these two?  GR is a fairly large 
> (100kDa homodimer) and is very stable, so I can only guess that
> the tag is somehow disturbed things at either the transcriptional
> or translational level-- rather than not allow the protein to fold
> and degrading.  I guess this is obvious, but any suggestions wou
> be appreciated.
> 
> Thanks.
> 
> 
> -------------------------------------------------------------------------------
> C. D. Walentas
> Cambridge Centre for Molecular Recognition,
> University of Cambridge,
> Cambridge, England CB2 1QW
> 
> Phone:  0223 333 662
> Fax:    0223 333 345
> email:  c.d.walentas at bioc.cam.ac.uk
> 
> 
> 



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