title: A two-plasmid system for independent genetic manipulation of subunits of homodimeric proteins and selective isolation of chimeric dimers.
abstract: We have designed and tested a modular two-plasmid expression system which allows coexpression of two different subunits of recombinant dimeric protein in Escherichia coli and selective purification of heterodimers. We have constructed a new expression vector, pBIOEx, with p15a replication origin which allows its stable coexistence with different ColE1 group plasmids. The expression cassette of this plasmid under control of the T7 promoter contains cloning site, followed by a short sequence coding for the C-terminal extension of the recombinant protein which is a target of the in vivo biotinylation by BirA protein. The expression unit is bicistronic, the second expressed protein being BirA. We have used this plasmid together with pET30a to clone kinesin heavy-chain fragment and coexpressed the two polypeptide chains differing by tags on their C-termini and we purified heterodimers made of two recombinant molecules. The heterodimeric protein had a normal biochemical activity. There was no discrimination against heterodimer formation at the dimerization step. The system is a powerful tool in studies of different aspects of interactions between subunits of the homodimeric proteins since it makes possible separate genetic manipulations on each subunit of the dimer.
authors.affiliation: Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland. kskowro@nencki.gov.pl
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Skowronek
authors.forename: Andrzej A
authors.initials: AA
authors.lastname: Kasprzak
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D019281:Dimerization; D004926:Escherichia coli; D015870:Gene Expression; D005818:Genetic Engineering; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D010957:Plasmids / Q000235:genetics*; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013552:Swine; D014404:Tubulin / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D000251:Adenosine Triphosphatases; D016547:Kinesins