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

J Biochem. 2009 Apr;145(4):533-42. doi: 10.1093/jb/mvp006. Epub 2009 Jan 17.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • CHO Cells
  • Consensus Sequence*
  • Cricetinae
  • Cricetulus
  • Electrophoresis, Polyacrylamide Gel
  • Glycosylation / drug effects
  • Humans
  • Mercaptoethanol / pharmacology
  • Plasmids / genetics*
  • Receptors, Complement / chemistry*
  • Receptors, Complement / genetics
  • Receptors, Complement / isolation & purification*
  • Repetitive Sequences, Amino Acid*
  • Subcellular Fractions / drug effects
  • Subcellular Fractions / metabolism

Substances

  • Receptors, Complement
  • Mercaptoethanol