pGEX expression problems

DENNIS P. SMITH SMITH_DENNIS_P at LILLY.COM
Tue Jun 22 21:24:17 EST 1993


We are using the vector pGEX-2T to express our protein as a fusion protein.
After induction with ITPG we get high level expression. The E.coli cell pellet
is respended in PBS with Triton X100, sonicated, and Sepharose-Glutathione
beads are added. The beads are washed with PBS and a sample of the beads are
taken for protein gel analysis. The fusion protein is eluted from the beads
using glutathione and a sample of the eluted protein taken for protein gel 
analysis. 

The question is after protein gel analysis we observe a single fusion band in
the induced cells but a doublet band in washed beads and a doublet for the
fusion protein eluted from the beads. Has anyone had this problem? We also
cannot digest the fusion protein with thrombin to release GST and our protein.
Has anyone had problems digesting with thrombin? 
			Dennis P. Smith
			smith_dennis_p at lilly.com

From: SMITH DENNIS P                (MCVAX0::BOOKS)

To:   VMS MAIL ADDRESSEE            (INT::"methods at genbank.bio.net")



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