Article

A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli.

Department of Biochemistry and Molecular Biology, Center for Gene Regulation, University Park, Pennsylvania 16802-1014, USA.
Protein Expression and Purification (Impact Factor: 1.43). 03/2001; 21(1):224-34. DOI: 10.1006/prep.2000.1363
Source: PubMed

ABSTRACT To facilitate studies of multicomponent protein complexes, I have developed an Escherichia coli expression system which coexpresses up to four polypeptides from a single plasmid. The modular nature of the system enables efficient subcloning of a gene into each of the 4 cassettes in the polycistronic expression vector. Restriction sites present in the polycistronic expression vector allow both affinity tagged and untagged complexes to be overexpressed. I demonstrate successful use of the expression system for binary and ternary complexes, including the reconstitution of the VHL-elonginC-elonginB complex in E. coli and purification of the complex by affinity and ion-exchange chromatography. This polycistronic expression system should provide an important alternative to in vitro reconstitution of multicomponent complexes.

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