Detection The Immunobiological Functions Of Effective Domain Of Recombinant Calreticulin

Calreticulin （CRT） is a ubiquitous and major Ca²⁺-binding protein, primarilyresident in the endoplasmic reticulum （ER） of eukaryotic cells, and it plays a centralrole in intracellular Ca²⁺homeostasis[1,2]. CRT is of46kDa with three domains followedby a four-amino acid ER retention sequence KDEL at the C terminus[1,2]. Thelectin-like globular N domain （amino acid residues18-197） of CRT is followed by aproline-rich P domain （residues198-308） and a Ca²⁺-binding C domain[1,3-5]. Similar tothe heat shock proteins（HSPs）, CRT is also a molecular chaperone in the ER, capable offacilitating peptide loading and correct folding of MHC class I molecules[2,5,6].Although CRT mainly localizes in intracellular compartments, it can also appear atthe surface of various types of cells and plays roles in cell-cell interaction, cell-matrixinteraction, and cell spreading[6-8]. CRT has been found on the membrane surface ofalmost all types of immune cells, including monocytes/macrophages,neutrophils,Tcells,and dendritic cells, exhibiting important functions in cell activation and clearanceof apoptotic and tumor cells[9-12]. Exposure of CRT on the membrane surface of tumorcells and apoptotic cells substantially increases their immunogenicity, rendering thecells recognized and attacked by phagocytosis more efficiently[13-16].However,molecular mechanisms for this phenomenon are not fully understood. Wereason that the immunobiological functions of CRT are most likely attributable to its Nrather than C domain, as the former is a lectin and capable of relatively high-affinitybinding with various glycosylated protein molecules[3-5].To understand the immunobiological functions of effective domain of CRT,Weexpress and purificate the murine recombinant CRT in E.coli. including rCRT/Ndomain, rCRT/120-250, rCRT/120-308, rCRT/251-360, rCRT/C domain. rCRT/Ndomain, rCRT/120-250, rCRT/120-308were purified using His6-tag （rCRT/N domainwas purified from the inclusion body protein）, rCRT/251-360and rCRT/C domain werepurified using GST-tag. When purifing the recombinant proteins，we find the concentration of rCRT/Ndomain is low.The purity of rCRT/251-360and rCRT/C domain are bad and the proteinsare not stable.From native-page analysis，we know rCRT/N domain，rCRT/251-360and rCRT/Cdomain can’t form dimer and polymer structures. rCRT/120-250and rCRT/120-308canstimulate the C57BL/6mouse spleen cells to secrete IL-6and induce the C57BL/6mouse peritoneal macrophages to secrete NO and TNF α. Immunogenicity assay of therecombinant proteins in BALB/c mice show rCRT/120-250and rCRT/120-308caninduce the higher titer of specific antibody production.Taken together， rCRT/120-250is the effective domain of FL-CRT. Theimmunobiological functions and immunogenicity of rCRT/120-250may be related withthe dimer and polymer structure.