title: A series of bacterial co-expression vectors with rare-cutter recognition sequences.
abstract: The bacterial co-expression method is a useful protein expression technique to reconstitute a hetero-oligomeric protein complex in vitro. However, during the plasmid subcloning for co-expression, unintended cleavage at the sequences of target cDNAs becomes more frequent as the number of DNA inserts increases. This problem also makes it difficult to change the combination of targeted proteins after preparing a certain co-expression construct. To avoid this problem, we have developed a series of bacterial co-expression vectors, in which each translation cassette can be subcloned at a set of rare-cutter restriction enzyme sites. We selected 9 different rare-cutter restriction enzymes that recognize a 7 or 8-base-pair sequence, and constructed 27 kinds of cloning vectors and 3 kinds of co-expression vectors, utilizing the rare-cutter recognition sequences as multi-cloning sites. Using this vector system, we co-expressed and co-purified a 7-subunit protein complex composed of the mammalian 26S proteasome regulatory subunits RPT1 to RPT6, and their associated factor, gankyrin. We verified the presence of all 7 subunits by western blotting, by taking advantage of the vector system in which the target proteins can be fused with a broad repertoire of epitope tags.
authors.affiliation: RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Wakamori
authors.forename: Takashi
authors.initials: T
authors.lastname: Umehara
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex / Q000235:genetics* / Q000302:isolation & purification; D021122:Protein Subunits / Q000235:genetics / Q000302:isolation & purification; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000302:isolation & purification
chemical_list: C403591:PSMD10 protein, human; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease