(426r) Cell-Free Production of Proteins Requiring Disulfide Bonds

     Cell-free Protein Synthesis (CFPS) is emerging as an
important technology for the production of proteins for structural and
functional analysis.  These post-genomic challenges require a CFPS system that can
incorporate post-translational modifications, including the formation of
disulfide bonds.  The cytoplasm of E. coli, and therefore a CFPS
reaction using E. coli extract, is kept reduced by two parallel pathways
mediated by the enzymes glutathione reductase (GR) and thioredoxin reductase
(TR).  Proteins requiring disulfide bonds can currently be produced in CFPS by derivatizing
the active site cysteines of GR and TR with iodoacetamide, thereby inactivating
them.  Unfortunately, the iodoacetamide reacts indiscriminately with all
reduced sulfhydryl groups in the extract.  This leads to a decrease in the
protein synthesis yields of these reactions and also inactivates a key enzyme
which precludes the use of glucose as an energy source in the CFPS reactions. 

     We will discuss the development of an E. coli strain,
and therefore an E. coli extract, that allows the formation of disulfide
bonds in CFPS reactions without using iodoacetamide.  The gene encoding the GR
enzyme has been deleted from the chromosome.  If both GR and TR are deleted, a
peroxiredoxin enzyme mutates to become a disulfide reductase.  The gene for TR has
therefore not been deleted, but has been modified to include an HA (hemagglutinin)
affinity tag.  The tagged TR is still active when the cells are grown, but can
be removed using an anti-HA column prior to using the extract in a CFPS
reaction.  We will present cell-free synthesis data for active Tissue Plasminogen
Activator (tPA), Urokinase, and Granulocyte Macrophage Colony Stimulating
Factor (GMCSF) requiring 9, 6, and 2 disulfide bonds respectively.  CFPS of
proteins requiring disulfide bonds using glucose as an economical energy source
will also be presented.  This technique should be generally applicable to other
situations where deleterious enzymes must be removed prior to CFPS, but can not
be deleted from the chromosome.