Journal of Biochemistry Advance Access originally published online on January 17, 2009
Journal of Biochemistry 2009 145(4):533-542; doi:10.1093/jb/mvp006
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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
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; and 3Division of Organ Transplantation, Department of Molecular Therapeutics, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871 Japan
To whom correspondence should be addressed. Tel: +81 29 853 7098, Fax: +81 29 853 7098, E-mail: cogitate{at}sakura.cc.tsukuba.ac.jp
Correspondence may also be addressed to Noriyuki Ishii. Tel: +81 29 861 6195, Fax: +81 29 861 6195, E-mail: ishii{at}ni.aist.go.jp
Received November 20, 2008; Accepted January 7, 2009
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Abstract |
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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 over-expression 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 
-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 haemolytic 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
Abbreviations: CHO, Chinese hamster ovary; CR1, complement receptor type 1; DHFR, dihydrofolate reductase; dhfr, dihydrofolate reductase gene; MAC, membrane attack complex; MTX, methotrexate; NaBu, sodium butylate; NHS, normal human serum; NMR, nuclear magnetic resonance; PNGase F, peptide-N4-(acetyl-β-glucosaminyl)-asparagine amidase; SCR, short consensus repeat; sCR1, soluble form of CR1; SCR1-3, the first three SCR modules from N-terminus of CR1; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; SRBC, sheep red blood cells
*These authors are equal contributors 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.