GST Fusion protein

TREACYB at NCCCOT.AGR.CA TREACYB at NCCCOT.AGR.CA
Mon Jun 24 15:41:22 EST 1996


  Dear Collegues,


  I'm using the prokaryotic GST fusion vector in hopes of purifying
a pollen-specific protein. I have had great success using the CDNB
detection module to work out my parameters and conditions. I get
great induction after growing overnight at 28C with 2% glucose..
followed by a 1:10 dilution into fresh YT with 2% glucose till cultures
are quite milky (A600 about 1.5) since my proteins appears to be toxic
as noticed by reduced size of pellet when NOT following these conditions.

  I induce by spinning down cultures and resuspending in fresh YT
(ampicillin always) but NO glucose and 0.1mM IPTG....or no IPTG but
then let culture go overnight again. CNDB assays are great....but
when I run on SDS PAGE...my fusion protein (expected 46 kDa) lane
is identical to my native GST plasmid control (26 kDa) !!!! No
observable 20 kDa + 26 Kda either....just a very intense 26KDa. The
GST-Bnm1 junction has been sequenced....The ATG IS IN FRAME with
GST!!! Can anyone help me? Or has anyone come across such a phenomenon?

  Thanks for your time..... Brian  Treacy





More information about the Methods mailing list