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.
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.
Yamaguchi
Atsushi
A
Biological Information Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Central-6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
Takagawa
Hiroaki
H
Iwakaji
Hirofumi
H
Miyagawa
Shuji
S
Wang
Pi-Chao
PC
Ishii
Noriyuki
N
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
D011951:Receptors, Complement; D008623:Mercaptoethanol