Expression, purification and characterization of TMCO1 for structural studies

Ningning Zhang; Meng Tang; Maorong Wen; Yu Cao; Bo OuYang

doi:10.1016/j.pep.2020.105803

Expression, purification and characterization of TMCO1 for structural studies

Protein Expr Purif. 2021 Mar:179:105803. doi: 10.1016/j.pep.2020.105803. Epub 2020 Nov 27.

Authors

Ningning Zhang¹, Meng Tang¹, Maorong Wen², Yu Cao³, Bo OuYang⁴

Affiliations

¹ State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 201203, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
² State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 201203, China.
³ Department of Orthopaedics and Institute of Precision Medicine, Shanghai Key Laboratory of Orthopaedic Implant, The Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, 115 Jinzun Road, Shanghai, 200125, China. Electronic address: yu.cao@shsmu.edu.cn.
⁴ State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 201203, China; University of Chinese Academy of Sciences, Beijing, 100049, China. Electronic address: ouyang@sibcb.ac.cn.

PMID: 33253810
DOI: 10.1016/j.pep.2020.105803

Abstract

Transmembrane and coiled-coil domains 1 (TMCO1) has a highly conserved amino acid sequence among species, indicating a critical role of TMCO1 in cell physiology. The deficiency of TMCO1 in humans is associated with cerebrofaciothoracic dysplasia (CFTD), glaucoma, osteogenesis and the occurrence of cancer. TMCO1 was recently identified as an endoplasmic reticulum (ER) Ca²⁺ load-activated Ca²⁺ (CLAC) release channel, which prevents ER Ca²⁺ overload and maintains calcium homeostasis in the ER. However, the structural basis of the molecular function of TMCO1 channel remains elusive. To determine the structure of TMCO1, we screened the expression of TMCO1 in Escherichia coli and insect cell expression systems. TMCO1 from Dictyostelium discoideum (DdTMCO1) was successfully expressed in Escherichia coli with a high yield. The pure recombinant protein was obtained by affinity chromatography and size exclusion chromatography. The solution NMR of DdTMCO1 in DPC micelles showed three α-helical transmembrane regions.

Keywords: Membrane protein expression; Micelles; Solution NMR; TMCO1.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Calcium Channels* / chemistry
Calcium Channels* / genetics
Calcium Channels* / isolation & purification
Calcium Channels* / metabolism
Dictyostelium / genetics
Escherichia coli / genetics
Humans
Nuclear Magnetic Resonance, Biomolecular
Protozoan Proteins / chemistry
Protozoan Proteins / genetics
Protozoan Proteins / isolation & purification
Protozoan Proteins / metabolism
Recombinant Proteins* / chemistry
Recombinant Proteins* / genetics
Recombinant Proteins* / isolation & purification
Recombinant Proteins* / metabolism
Sf9 Cells

Substances

Calcium Channels
Protozoan Proteins
Recombinant Proteins
TMCO1 protein, human