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Journal of Biochemistry Advance Access published online on January 17, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp006
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Construction of the Plasmid, Expression by Chinese Hamster Ovary Cell, Purification, and Characterization of the First Three Short Consensus Repeat Modules of Human Complement Receptor Type 1

Atsushi Yamaguchi1,2,*, Hiroaki Takagawa1,2,*, Hirofumi Iwakaji2, Shuji Miyagawa3, Pi-Chao Wang1,2,{dagger} and Noriyuki Ishii1,$,{dagger}

1Biological Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central-6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566; 2Gradute School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572; 3Division of Organ Transplantation, Department of Molecular Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871 Japan

{dagger}To whom correspondence should be addressed: Noriyuki Ishii, Tel/Fax: +81 29 853 7098, E-mail: cogitate{at}sakura.cc.tsukuba.ac.jp (P.-C. Wang), Tel/Fax: +81 29 861 6195, E-mail: ishii{at}ni.aist.go.jp (N. Ishii)

Received November 20, 2008; Accepted January 7, 2009


  Abstract

Short consensus repeat (SCR1-3), the first three SCR modules from N-terminus of type 1 complement receptor (CR1), is expected to accelerate dissociation of complement components and suppress complement activity by binding the main component of complement C4b. In order to clarify the three dimensional structure which triggers the activity of SCR1-3 on complement, we constructed an overexpression system in CHO DG44 cells which facilitated mass production of SCR1-3. The mass production was achieved by a two-stage culture system and optimum culture conditions using ASF104N medium and MTX-, NaBu-containing {alpha}-MEM/10% FBS medium, respectively. The constructed gene of SCR1-3 was confirmed by restriction enzyme digestion and DNA sequence analysis, and the expressed protein by CHO DG44 cells was confirmed by Western blotting. The expressed SCR1-3 was proved containing N-linked sugar chain, an important factor to the proper expression of protein, by the cleavage with glycosidase of N-linked oligosaccharide (PNGase F). The suppression effect of the yield protein on complement-mediated inflammation was investigated by hemolytic assay and necrosis assay of stromal cells. Both assays showed that SCR1-3 possessed complement control activity. However, residing sugar chain on SCR1-3 did not show significant difference in the complement control activity.

Key Words: Complement control activity, Complement receptor type 1 (CR1), Short consensus repeat (SCR1-3), CHO cell, Sugar chain

*These authors contributed equally to this work.

$Present address: Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Central-6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan


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