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<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=401431606-09072003>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.</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=401431606-09072003>Practically, we observed high percentage of misfolded
and aggregated molecules or truncated translation products (sometimes
misinterpreted as "proteolytic degradation products").</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN class=401431606-09072003>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 ...</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN class=401431606-09072003>Best
regards,</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN
class=401431606-09072003>Hans-Joachim Schönfeld.</SPAN></FONT></DIV>
<DIV><FONT face=Arial color=#0000ff size=2><SPAN class=401431606-09072003><!-- Converted from text/rtf format -->
<P><SPAN lang=de-ch><FONT face=Tahoma
size=1>============================================</FONT></SPAN> <BR><SPAN
lang=de-ch><FONT face=Tahoma size=1>Dr. Hans-Joachim Schönfeld</FONT></SPAN>
<BR><SPAN lang=de-ch><FONT face=Tahoma size=1>F. Hoffmann-La Roche
Inc.</FONT></SPAN> <BR><SPAN lang=de-ch><FONT face=Tahoma size=1>PRBD-E,
B93/5.44</FONT></SPAN> <BR><SPAN lang=de-ch><FONT face=Tahoma size=1>CH-4070
Basel</FONT></SPAN> <BR><SPAN lang=de-ch><FONT face=Tahoma
size=1>Switzerland</FONT></SPAN> </P>
<P><SPAN lang=de-ch><FONT face=Tahoma size=1>Tel. (+41) 61 688 28
95</FONT></SPAN> <BR><SPAN lang=de-ch><FONT face=Tahoma size=1>Fax. (+41) 61 688
90 60</FONT></SPAN> <BR><SPAN lang=de-ch><FONT face=Tahoma size=1><A
href="mailto:hans-j.schoenfeld@roche.com">mailto:hans-j.schoenfeld@roche.com</A></FONT></SPAN><SPAN
lang=en-us></SPAN><SPAN lang=en-us></SPAN><SPAN lang=en-us></SPAN>
</P></SPAN></FONT></DIV></DIV>
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<DIV></DIV>
<DIV class=OutlookMessageHeader lang=en-us dir=ltr align=left><FONT
face=Tahoma size=2>-----Original Message-----<BR><B>From:</B>
rasmb-admin@rasmb-email.bbri.org [mailto:rasmb-admin@rasmb-email.bbri.org]
<B>On Behalf Of </B>John Rodgers<BR><B>Sent:</B> Wednesday, July 09, 2003
12:11 AM<BR><B>To:</B> 'RASMB@rasmb-email.bbri.org'<BR><B>Subject:</B> [RASMB]
His tagged proteins<BR><BR></FONT></DIV>
<DIV><FONT face=Arial size=2>
<DIV><SPAN class=112312921-08072003><FONT face=Arial size=2>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. </FONT></SPAN></DIV>
<DIV><SPAN class=112312921-08072003><FONT face=Arial
size=2>Thanks,</FONT></SPAN></DIV>
<DIV><SPAN class=112312921-08072003><FONT face=Arial
size=2>John Rodgers </FONT></SPAN></DIV></FONT></DIV></BLOCKQUOTE></BODY></HTML>