title: Effects of E. coli chaperones on the solubility of human receptors in an in vitro expression system.
abstract: The implementation of efficient technologies for the production of recombinant mammalian membrane receptors is an outstanding challenge in understanding receptor-ligand actions and the development of therapeutic antibodies. In order to improve the solubility of recombinant extracellular domains of human membrane receptors expressed in Escherichia coli, proteins were synthesized by an E. coli in vitro translation system supplemented with bacterial molecular chaperones, such as GroEL-GroES (GroEL/ES), Trigger factor (TF), a DnaK-DnaJ-GrpE chaperone system (DnaKJE), and/or a heat shock protein Hsp100, ClpB. The following three proteins that are prone to aggregation were examined: the extracellular domain (ECD) or the second immunoglobulin-like domain (IgII) of the human neurotrophin receptor TrkC (TrkC-ECD and TrkC-IgII), and the C-type lectin carbohydrate recognition domain of the human asialoglycoprotein receptor (ASGPR HI CRD). The cooperative chaperone system including GroEL/ES, DnaKJE and ClpB had a marked effect on the solubility of TrkC-ECD and TrkC-IgII, and the GroEL/ES-DnaKJE-TF chaperone system was more effective for TrkC-IgII. The GroEL/ES-DnaKJE-TF chaperone network increased the yield of soluble ASGPR HI CRD. The present findings demonstrate that E. coli molecular chaperones are useful in improving the yield of soluble recombinant extracellular domains of human membrane receptors in an E. coli expression system.
authors.affiliation: Biological Science Laboratories, Toray Research Center, Inc., 1111 Tebiro, Kamakura, Kanagawa 248-8555, Japan.
authors.forename: Sumiyo
authors.initials: S
authors.lastname: Endo
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Tomimoto
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Shimizu
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Taniguchi
authors.forename: Takuo
authors.initials: T
authors.lastname: Onizuka
mesh_terms: D002474:Cell-Free System / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D011956:Receptors, Cell Surface / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012995:Solubility
chemical_list: D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins