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.
Copyright 2001 Academic Press.
MeSH terms
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Base Sequence
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Chromatography, Affinity / methods
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Chromatography, Ion Exchange / methods
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Cloning, Molecular / methods*
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DNA Primers
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DNA, Fungal / genetics
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Elongin
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Escherichia coli / genetics*
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Fungal Proteins / genetics
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Fungal Proteins / isolation & purification
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Genes
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Genetic Vectors
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Humans
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Ligases*
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Macromolecular Substances
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Plasmids
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Polymerase Chain Reaction
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Proteins / genetics*
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Proteins / isolation & purification*
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Recombinant Proteins / biosynthesis*
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Recombinant Proteins / isolation & purification
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Restriction Mapping
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Saccharomyces cerevisiae / genetics
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Transcription Factors / genetics*
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Transcription Factors / isolation & purification*
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Tumor Suppressor Proteins*
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Ubiquitin-Protein Ligases*
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Von Hippel-Lindau Tumor Suppressor Protein
Substances
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DNA Primers
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DNA, Fungal
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Elongin
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Fungal Proteins
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Macromolecular Substances
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Proteins
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Recombinant Proteins
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Transcription Factors
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Tumor Suppressor Proteins
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Ubiquitin-Protein Ligases
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Von Hippel-Lindau Tumor Suppressor Protein
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Ligases
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VHL protein, human