W0388

Producing Protein Complexes in Escherchia coli by Polycistronic Expression. Song Tan, Will Selleck, Jordan Lichty, Joshua Malecki, Adam Barrios, Brian Hnatkovich, Biochemistry & Molecular Biology, Penn State Univ., 108 Althouse Lab, University Park, PA 16802 USA.

A major barrier to the structural determination of multicomponent protein complexes is isolation of sufficient material for crystallization studies. We have developed an E. coli expression system which coexpresses up to four polypeptides from a single plasmid to tackle this problem. The modular nature of the system enables (a) efficient subcloning of a gene into each of the four cassettes in the polycistronic expression vector and (b) simple incorporation of both N- and C-terminal afffinity tags. We have successfully used this expression system to reconstitute and purify milligram quantities of several two, three and four subunit yeast or human protein complexes with components that could not be solubly expressed on their own. For example, we have isolated and are pursuing the crystallization of a catalytic subcomplex of the yeast SAGA histone acetyltransferase complex containing 50, 51 and 80 kD polypeptides.