A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli.
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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Department of Biochemistry and Molecular Biology, Center for Gene Regulation, University Park, Pennsylvania 16802-1014, USA.
D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004271:DNA, Fungal / Q000235:genetics; D000075065:Elongin; D004926:Escherichia coli / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D008025:Ligases; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification*; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein
D017931:DNA Primers; D004271:DNA, Fungal; D000075065:Elongin; D005656:Fungal Proteins; D046911:Macromolecular Substances; D011506:Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; D051794:Von Hippel-Lindau Tumor Suppressor Protein; D008025:Ligases; C497446:VHL protein, human