Protein-protein interaction of His-tag of pET14b?

Jimmy S.H. Tsang jshtsang at hkucc.hku.hk
Thu Mar 6 19:20:12 EST 1997


In article <E6H12n.IC6 at fsa.bris.ac.uk>, bijgh at zeus.bris.ac.uk (Jared Head) wrote:
>Jimmy S.H. Tsang (jshtsang at hkucc.hku.hk) wrote:
>: 
>: Dear All,
>:         I have recently fused a bacterial gene downstream of the His-tag 
>: sequence of the pET14b from AMS Biotech.  The in vitro transcribed and 
>: translated protein forms a complex which migrates much slower than the wild 
>: type protein in a native PAGE.  Did anyone experience a similar 
>: 'multimerisation' problem?  The same gene cloned into pET19b produced a 
>: protein similar to that of native protein, i.e. no 'multimerisation'.  Any 
>: suggestion and comment will be appreciated.
>
>We're just finding the same thing.  It seems possible that more than one
>protein are chelating around one nickel atom.  When we cleaved the His.Tag
>off we made our protein monomeric, and we also found dtt to have the same
>effect, presumably by reducing the nickel ions.
>
>Jared
>
>-- 
>Jared Head     at the Department of Biochemistry, University of Bristol

Dear Jared,
        Thank you for the reply.  However in our hands we have already use DTT 
to stabilise our proteins during the running of the gel.  Our native PAGE gel 
does not contain additional nickel so binding of more than one protein complex 
around the nickel atom seems unlikely.  Our native protein is already a 
homodimer but expression from pET14b produce more than a dimer and no dimeric 
protein fraction was detected.  
        We don't think the 'multimerisation' was caused by the His-Tag because 
expression from pET19b which also contain the His-Tag did not produce such an 
effect.
        Thank you ones again for all your comment.

J.S.H. Tsang
Department of Botany
The University of Hong Kong
jshtsang at hkucc.hku.hk
http://www.hku.hk/botany/staff/tsang.html



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