N-Linked Glycan Characterization of Heterologous Proteins

Li, Huijuan; Miele, Robert G.; Mitchell, Teresa; Gerngross, Tillman U.

doi:10.1385/1-59745-456-7:139

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“…The flow rate was 1.0 ml/min for 40 min and the column was maintained to 30 °C. After eluting isocratically (68% A:32% B) for 3 min, a linear solvent gradient (68% A:32% B to 40% A:60% B) was employed over 27 min to elute the glycans (Li et al , 2007). Solvent A (acetonitrile) and solvent B (ammonium formate, 50 m M , pH 4.5).…”

Section: Methodsmentioning

confidence: 99%

A combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris

Nett¹,

Stadheim²,

Li³

et al. 2011

Yeast

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To humanize the glycosylation pathway in the yeast Pichia pastoris, we developed several combinatorial genetic libraries and used them to properly localize active eukaryotic mannosidases and sugar transferases. Here we report the details of the fusion of up to 66 N -terminal targeting sequences of fungal type II membrane proteins to 33 catalytic domains of heterologous glycosylation enzymes. We show that while it is difficult to predict which leader/catalytic domain will result in the desired activity, analysis of the fusion protein libraries allows for the selection of the leader/catalytic domain combinations that function properly. This combinatorial approach, together with a high-throughput screening protocol, has allowed us to humanize the yeast glycosylation pathway to secrete human glycoprotein with complex N -glycosylation.

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Section: Methodsmentioning

confidence: 99%