title: EcoExpress-Highly Efficient Construction and Expression of Multicomponent Protein Complexes in Escherichia coli.
abstract: The bacterium Escherichia coli remains the leading host for protein expression in large quantity for the purpose of crystallization or other biochemical studies. However, expression of multicomponent protein complexes remains a challenge, and is often laborious and time-consuming. Here we developed a method named EcoExpress, which allows efficient construction of plasmids to express individual protein with user-defined epitope-tag, followed by one-pot assembly of a single vector to express the entire protein complex for copurification. A versatile set of vectors was designed to provide various choices to control the expression of a protein with different promoters, and to accept different number of components for coexpression. Using EcoExpress, we demonstrated that each subunit within a protein complex could be expressed individually or simultaneously, and the entire complex could be copurified. In addition, to overcome the decreased assembly efficiency with the increasing number of components, a novel oligonucleotides blocking method was designed and tested. EcoExpress provides the scientific community with a toolbox to rapidly investigate the function of protein complexes.
authors.affiliation: MOE Key Laboratory of Bioinformatics, MOE Key Laboratory of Industrial Biocatalysis and Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University , Beijing 100084, China.
authors.forename: Yiran
authors.initials: Y
authors.lastname: Qin
authors.affiliation: MOE Key Laboratory of Bioinformatics, MOE Key Laboratory of Industrial Biocatalysis and Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University , Beijing 100084, China.
authors.forename: Chang
authors.initials: C
authors.lastname: Tan
authors.affiliation: Wuxi Qinglan Biotechnology Inc. , Yixing, Jiangsu 214200, China.
authors.forename: Jiwei
authors.initials: J
authors.lastname: Lin
authors.affiliation: MOE Key Laboratory of Bioinformatics, MOE Key Laboratory of Industrial Biocatalysis and Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University , Beijing 100084, China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Qin
authors.affiliation: MOE Key Laboratory of Bioinformatics, MOE Key Laboratory of Industrial Biocatalysis and Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University , Beijing 100084, China.
authors.forename: Jianghaiyang
authors.initials: J
authors.lastname: He
authors.affiliation: MOE Key Laboratory of Bioinformatics, MOE Key Laboratory of Industrial Biocatalysis and Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University , Beijing 100084, China.
authors.forename: Qingyu
authors.initials: Q
authors.lastname: Wu
authors.affiliation: School of Biological Sciences, The King's Buildings, University of Edinburgh , EH9 3BF Edinburgh, United Kingdom.
authors.forename: Yizhi
authors.initials: Y
authors.lastname: Cai
authors.affiliation: MOE Key Laboratory of Bioinformatics, MOE Key Laboratory of Industrial Biocatalysis and Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University , Beijing 100084, China.
authors.forename: Zhucheng
authors.initials: Z
authors.lastname: Chen
authors.affiliation: MOE Key Laboratory of Bioinformatics, MOE Key Laboratory of Industrial Biocatalysis and Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University , Beijing 100084, China.
authors.forename: Junbiao
authors.initials: J
authors.lastname: Dai
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D053938:DNA Fragmentation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D056968:Lac Repressors / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic
chemical_list: D001426:Bacterial Proteins; C068356:CcdB protein, Plasmid F; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D046912:Multiprotein Complexes
keywords: cloning method; coexpression; protein complex; protein expression; synthetic biology; vector system