[RASMB] His tagged proteins

[Schoenfeld, Hans-J. {PRBD~Basel}]

Pragmatically: a tagged protein is not authentic regarding the primary structure of the natural protein - why should it be regarding its quaternary structure? In particular the hexahis tag places unique biophysical properties on any protein molecule (successive hydrophobic or positively charged residues, dependent on the ph). This may be even more critical, when the tag is placed on the N-terminus of the protein chain, as protein translation starts from the N-terminus and folding occurs co-translational, just beginning after expression of some 20 N-terminal residues.
Practically, we observed high percentage of misfolded and aggregated molecules or truncated translation products (sometimes misinterpreted as "proteolytic degradation products").
For the above reasons I recommend the following. Start work with authentic protein sequence. Using state of the art separation technologies, purification of overexpressed recombinant proteins is straight forward, as far as the target protein is not aggregated but correctly folded. If you have to use a tag for some reasons, place it at the C-terminus. This prevents truncated molecules and folding may be in a progressed state before the tag gets translated. Always check preparations of purified protein by light scattering techniques (static or dynamic) or/and SEC, which are fast, before starting the time consuming but more detailed analysis by AUC ...
Best regards,
Hans-Joachim Schönfeld.
============================================ 
Dr. Hans-Joachim Schönfeld 
F. Hoffmann-La Roche Inc. 
PRBD-E, B93/5.44 
CH-4070 Basel 
Switzerland 

Tel. (+41) 61 688 28 95 
Fax. (+41) 61 688 90 60 
mailto:hans-j.schoenfeld at roche.com 

	-----Original Message-----
	From: rasmb-admin at rasmb-email.bbri.org [mailto:rasmb-admin at rasmb-email.bbri.org] On Behalf Of John Rodgers
	Sent: Wednesday, July 09, 2003 12:11 AM
	To: 'RASMB at rasmb-email.bbri.org'
	Subject: [RASMB] His tagged proteins
	
	
	
	I can imagine problems with performing ultracentrifugation experiments with His tagged proteins.  It seems that this was also mentioned in the recent workshop. A set of equilibrium runs were made before the workshop with the more abundant and available His tagged protein provided by the researcher.  With regard to the protein,  SEC static light scattering experiments were performed on both native extracted proteins and His tagged protein with consistent results (dimer-?tetramer in rapid equilibrim.).    The researcher makes the argument that the His tags are of no significant consequence, but is open to doing the extra work if a good case can be made for doing so.  Experience, advice and references appreciated.  
	Thanks,
	John Rodgers  

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