One-step affinity purification of fetuin from fetal bovine serum.
Fetuin is a plasma glycoprotein widely distributed in mammals. It has been used as a model protein for structural analyses and investigations into the biological properties of glycoproteins. A convenient one-step procedure for biospecific isolation of fetuin from fetal bovine serum was developed on the basis of wheatgerm agglutinin (WGA) affinity separation. Two different porous supports, a silica-based material and a polymer-based material, were used for the immobilization of WGA. The prepared WGA adsorbents were characterized and process parameters of the affinity separation of fetuin were investigated and optimized. WGA was immobilized on silica and polymer supports with coupling yields of 99.6 and 99.4% respectively and amounts of coupled ligand of 7.9 and 9.2 mg of WGA/ml respectively. It has been shown that the specific capacities for fetuin were 5.1 mg/ml on WGA-silica, 1.8 mg/ml on WGA-polymer and 4.1 mg/ml on WGA-agarose. All three adsorbents proved to be suitable for the biospecific separation of fetuin. The polymer-based WGA adsorbent was successfully applied in the purification of fetuin from fetal bovine serum in a one-step separation process. The identity and purity of the isolated product was verified by SDS/PAGE. Under optimized conditions up to 21.6 mg of fetuin could be isolated from 1 ml of serum. The procedure described was designed to be easily scaled-up for the production of fetuin.
Cartellieri
Simone
S
GKSS National Research Center, Institute for Coastal Research/Physical and Chemical Analysis, Max-Planck-Strasse, D-21502 Geesthacht, Federal Republic of Germany.
Hamer
Ole
O
Helmholz
Heike
H
Niemeyer
Bernd
B
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D005260:Female; D005312:Fetal Blood; D011247:Pregnancy; D014909:Wheat Germ Agglutinins; D000509:alpha-Fetoproteins / Q000302:isolation & purification*
D014909:Wheat Germ Agglutinins; D000509:alpha-Fetoproteins