Effects of E. coli chaperones on the solubility of human receptors in an in vitro expression system |
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Authors: | Sumiyo Endo Yusuke Tomimoto Hiroyuki Shimizu Yoshitaka Taniguchi Takuo Onizuka |
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Institution: | (1) Biological Science Laboratories, Toray Research Center Inc., 1111 Tebiro, Kamakura, 248-8555 Kanagawa, Japan |
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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. |
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Keywords: | E coli in vitro chaperones GroEL/ES DnaKJE TF ClpB solubility |
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