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Glycosylationproperties of the Pichia pastoris ‐expressed recombinant kringle 2 domain oftissue‐type plasminogen activator

1997; Wiley; Volume: 25; Issue: 2 Linguagem: Inglês

10.1111/j.1470-8744.1997.tb00427.x

1470-8744

Robert G. Miele, Stephanie L. Nilsen, Teresa Brito, Roger K. Bretthauer, Francis Castellino,

Glycosylation and Glycoproteins Research

The oligosaccharide structures present on Asn 5 of the Pichia pastoris ‐expressed recombinant kringle 2 domain of tissue‐type plasminogen activator {(r)‐[K2 tPA ]} have been determined by a combination of techniques, including HPLC, FPLC, gel filtration, endoglycosidase digestions and mass spectrometry. The major oligosaccharides identified after their liberation by either hydrazinolysis or by the enzyme peptide: N 4 ‐( N ‐acetyl‐b‐glucosaminyl)asparaginyl amid‐ase, were in the oligomer range of (mannose) 8 ( N ‐acetylglucosamine) 2 (Man 8 GN 2 ) to Man 18 GN 2 . The preponderance of these glycans spanned Man 9 GN 2 to Man 12 GN 2 , and the major overall product was Man 10 GN 2 . An additional (less than 5′) amount of the polypeptide was hyperglycosylated. In contrast with glycoproteins produced in Saccharomyces cerevisiae , our results with specific mannosidase digestions were consistent with previous studies showing that (a1,3)‐linked mannose residues were not present in extensions of the core Man 8 GN 2 unit. The results show that the N‐linked glycosylation pathways in P. pastoris are substantially different from those found in S. cerevisiae , with shorter Man(a1,6) extensions to the core Man 8 GN 2 and the apparent lack of significant Man(a1,3) additions representing the major processing modality of N‐linked glycans in P. pastoris.