A simple two-step purification procedure for the iC3b binding collectin conglutinin.
Bovine conglutinin is a serum protein involved in innate immunity. It binds calcium dependently to iC3b, a product of the complement component C3 deposited on cell surfaces, immune complexes or artificial surfaces after complement activation. We here present a simple and efficient two-step procedure for the purification of conglutinin. In the first step, bovine serum is incubated with non-coupled chromatographic TSK beads at 37°C to allow complement activation and iC3b deposition on the beads and subsequent binding of conglutinin to iC3b. Conglutinin is then eluted from the beads by EDTA. In the second step, conglutinin is separated from iC3b and IgM by ion-exchange chromatography. This purification procedure yielded 81 μg of conglutinin per ml of serum with a recovery of 61.2%. Surface plasmon resonance analysis showed that the purified conglutinin had a high affinity for mannan (K(d)=2.3-3.2 nM). SDS-PAGE and time-resolved immunofluorometric assays showed that the conglutinin was not contaminated with other serum collectins such as collectin-43 or mannan-binding lectin.
Krogh-Meibom
Thomas
T
Department of Animal Health and Bioscience, Faculty of Agricultural Sciences, Aarhus University, 8830 Tjele, Denmark.
Ingvartsen
Klaus Lønne
KL
Tornoe
Ida
I
Palaniyar
Nades
N
Willis
Anthony C
AC
Holmskov
Uffe
U
D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D037222:Collectins / Q000737:chemistry / Q000302:isolation & purification*; D003167:Complement Activation; D003179:Complement C3b; D008351:Mannans / Q000737:chemistry; D012712:Serum Globulins / Q000737:chemistry / Q000302:isolation & purification*
D037222:Collectins; D008351:Mannans; D012712:Serum Globulins; C024014:conglutinin; D003179:Complement C3b