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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© 2007 Humana Press Inc., Totowa, NJ
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Li, H., Miele, R.G., Mitchell, T.I., Gerngross, T.U. (2007). N-Linked Glycan Characterization of Heterologous Proteins. In: Cregg, J.M. (eds) Pichia Protocols. Methods in Molecular Biology, vol 389. Humana Press. https://doi.org/10.1007/978-1-59745-456-8_10
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DOI: https://doi.org/10.1007/978-1-59745-456-8_10
Publisher Name: Humana Press
Print ISBN: 978-1-58829-429-6
Online ISBN: 978-1-59745-456-8
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