title: In vitro refolding of recombinant human free secretory component using equilibrium gradient dialysis.
abstract: Human secretory component (SC) is associated with secretory immunoglobulins (IgA and IgM) and serves to protect the immunoglobulin in the harsh mucosal environment. SC is derived from the polymeric immunoglobulin receptor (pIgR) which transports polymeric immunoglobulins across epithelial cells into secretions. In this present study, we describe the first cloning, expression, in vitro refolding and purification of a free form of human secretory component (rSC) containing the five functional ligand binding domains using Escherichia coli BL21 (DE3). Free rSC was refolded from inclusion bodies by equilibrium dialysis after purification by nickel affinity chromatography under denaturing conditions. Refolded rSC was purified by gel filtration chromatography. Surface plasmon resonance and dot blot association analysis have shown that purified rSC binds IgM with a physiological equilibrium dissociation constant (KD) of 4.6x10(-8) M and shares structural similarity to native SC. This provides an important step in the elucidation of the structure of this immunologically important receptor.
authors.affiliation: Department of Biochemistry and Microbiology, Faculty of Science, Nelson Mandela Metropolitan University South Campus, Summerstrand, P.O. Box 77000, Port Elizabeth 6031, South Africa.
authors.forename: Earl
authors.initials: E
authors.lastname: Prinsloo
authors.forename: Vaughan
authors.initials: V
authors.lastname: Oosthuizen
authors.forename: Koji
authors.initials: K
authors.lastname: Muramoto
authors.forename: Ryno J
authors.initials: RJ
authors.lastname: Naude
mesh_terms: D001666:Binding Sites, Antibody; D003956:Dialysis; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007075:Immunoglobulin M / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012635:Secretory Component / Q000235:genetics / Q000378:metabolism*
chemical_list: D007075:Immunoglobulin M; D011994:Recombinant Proteins; D012635:Secretory Component