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Journal of Biochemistry Advance Access originally published online on July 24, 2009
Journal of Biochemistry 2009 146(5):713-723; doi:10.1093/jb/mvp117
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Expression, Purification and Characterization of Soluble Recombinant Periostin Protein Produced by Escherichia coli

Issei Takayama1,2, Isao Kii1 and Akira Kudo1,*

1Department of Biological Information, Graduate School of Bioscience and Biotechnology; and 2Global COE Program, Evolving Education and Research Center for Spatio-Temporal Biological Network, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Midori-ku, Nagatsuta, Yokohama 226-8501, Japan

*To whom correspondence should be addressed. Tel/Fax: +81-45-924-5719, E-mail: akudo{at}bio.titech.ac.jp

Received June 20, 2009; Accepted July 11, 2009


  Abstract

Periostin is a matricellular protein participating in the tissue remodelling of damaged cardiac tissue after acute myocardial infarction and of the periodontal ligament in mice. However, further studies on the periostin protein have been limited by the intrinsic difficulty of purifying this protein produced in Escherichia coli due to its insolubility. Here, we demonstrate the expression of recombinant periostin protein with high solubility and monodispersity in E. coli. Periostin is composed of an amino-terminal EMI domain, a tandem repeat of 4 fas1 domains (RD1-4), and a carboxyl-terminal region (CTR). We expressed the RD4-CTR region tagged with GST at amino-terminal and 6x Histidine at carboxyl-terminal end in E. coli. The recombinant protein was purified by using GSH-Sepharose and nickel chelation affinity chromatography, followed by gel filtration chromatography. The RD4-CTR protein exhibited high solubility and monodispersity. On average, 9.1 mg of purified RD4-CTR was routinely obtained from 1 L of culture media. Furthermore, the RD4-CTR was biochemically active, because it bound to the RD1-4, the same as intact periostin protein that had been purified from mammalian cells. Our results should enable us to produce the periostin recombinant protein in large quantities and facilitate future studies on functional and structural analyses of periostin.

Key Words: extracellular matrix, fasciclin I, periodontal ligament, periosteum, periostin

Abbreviations: GST, glutathione-S-transferase; CBB, Coomassie brilliant blue; HRP, horseradish peroxidase; ECM, extracellular matrix; RD, repeat domain; RD1-4, tandem repeat of 4 fas1 domains; Ni-NTA, nickel chelation affinity chromatography; CTR, carboxyl-terminal region; DMEM, Dulbecco's modified eagle medium; FBS, fetal bovine serum, PBS, phosphate-buffered saline; HA, haemagglutinin; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid


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