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Biotechnology and Applied Biochemistry (2002) 35, (83–89) (Printed in Great Britain)

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One-step affinity purification of fetuin from fetal bovine serum

Simone Cartellieri*, Ole Hamer*, Heike Helmholz* and Bernd Niemeyer*†¹

*GKSS National Research Center, Institute for Coastal Research/Physical and Chemical Analysis, Max-Planck-Strasse, D-21502 Geesthacht, Federal Republic of Germany, and †University of Federal Armed Forces Hamburg, Institute of Thermodynamics, Holstenhofweg 85, D-22043 Hamburg, Federal Republic of Germany

Key words: affinity adsorbent, affinity separation, lectin, polymer, silica.

Abbreviations used: Silica, Silica XWP-I005; Toyopearl, Toyopearl AF-Tresyl-650 M; WGA, wheatgerm agglutinin; PBD, Protein Data Bank; GlcNAc, N-acetyl-D-glucosamine; NeuNAc, N-acetylneuraminic acid; FBS, fetal bovine serum.

¹To whom correspondence should be sent, at the following address: Institut für Küstenforschung/Physikalische und Chemische Analytik, GKSS-Forschungszentrum Geesthacht GmbH, Max-Planck-Strasse, D-21502 Geesthacht, Germany (e-mail bernd.niemeyer@gkss.de).

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.

Received 21 August 2001/1 December 2001; accepted 7 December 2001

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