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Biotechnology and Applied Biochemistry (1998) 28, (39–45) (Printed in Great Britain)

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O-Mannosylation of Pichia pastoris cellular and recombinant proteins

Joseph G. Duman*¹, Robert G. Miele*, Hong Liang†, Davida K. Grella*†, Kim Lee Sim†, Francis J. Castellino* and Roger K. Bretthauer*²

*Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, U.S.A., and †EntreMed, 9610 Medical Center Drive, Suite 200, Rockville, MD 20850, USA

¹Present address: Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, U.S.A.

²To whom correspondence should be addressed.

O-linked saccharides were released from a major cell wall glycoprotein and from cellular mannan–protein complexes obtained from Pichia pastoris cells. Analysis by a variety of chromatographic methods and exoglycosidase digestions revealed the presence of mannose and (a1-2)-linked dimer, trimer and tetramer saccharides of mannose. The recombinant kringle 1–4 domain of human plasminogen expressed in P. pastoris was subjected to hydrazinolysis of both O- and N-linked saccharides. Only a very small quantity of N-linked oligosaccharides was present on the Asn²⁸⁹ consensus site. The major products were O-linked (a1-2)-linked mannans, containing dimeric, trimeric, tetrameric and pentameric oligosaccharides with the major amount of the total saccharides being distributed approximately equally between the dimer and trimer components. These results show that short O-linked saccharides of mannose containing (a1-2) glycosidic linkages are present in P. pastoris cells and expressed proteins.

Received 26 February 1998; accepted 8 March 1998

Portland Press Ltd © 1998