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Updated: Jul 10, 2026

Bioinformatics Resources for the Study of Glycan-Mediated Protein Interactions
Published on: January 20, 2022
N-linked glycan characterization of heterologous proteins
Huijuan Li1, Robert G Miele, Teresa I Mitchell
1GlycoFi Inc., Lebanon, NH, USA.
Abstract:
Our laboratory has focused on the re-engineered of the secretory pathway of Pichia pastoris to perform glycosylation reactions that mimic processing of N-glycans in humans and other higher mammals (1,2). A reporter protein with a single N-linked glycosylation site, a His-tagged Kringle 3 domain of human plasminogen (K3), was used to identify combinations of optimal leader/catalytic domain(s) to recreate human N-glycan processing in the Pichia system. In this chapter we describe detailed protocols for high-throughput purification of K3, enzymatic release of N-glycans, matrix-assisted laser desorption ionization time-of-flight and high-performance liquid chromatography analysis of the released N-glycans. The developed protocols can be adapted to the characterization of N-glycans from any purified protein expressed in P. pastoris.
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