title: 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.
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 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 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 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.
authors.affiliation: Biological Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central-6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yamaguchi
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Takagawa
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Iwakaji
authors.forename: Shuji
authors.initials: S
authors.lastname: Miyagawa
authors.forename: Pi-Chao
authors.initials: PC
authors.lastname: Wang
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Ishii
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D016384:Consensus Sequence; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D008623:Mercaptoethanol / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics*; D011951:Receptors, Complement / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D020449:Repetitive Sequences, Amino Acid; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism
chemical_list: D011951:Receptors, Complement; D008623:Mercaptoethanol