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High-Throughput Multimodal Strong Anion Exchange Purification and N-Glycan Characterization of Endogenous Glycoprotein Expressed in Glycoengineered Pichia pastoris.
MedLine Citation:
PMID:  22735962     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
The secretory pathway of the yeast Pichia pastoris has been engineered to produce complex human-type N-glycans (Choi et al., Proc Natl Acad Sci USA 100:5022-5027, 2003; Hamilton et al., Science 301:1244-1246, 2003; Hamilton et al., Science 313:1441-1443, 2006). In contrast to the heterogeneous glycans produced on the therapeutic glycoproteins expressed in mammalian cell lines, glycoengineered P. pastoris can be designed to produce a specific, preselected glycoform. In order to achieve glycan uniformity on the target protein, No Open Reading Frame (NORF) yeast cell lines are screened extensively during various stages of glycoengineering. In the absence of the target protein of interest, screening the NORF yeast cell lines for glycoform uniformity becomes a challenge. The common approach so far has been to analyze the total cell glycan pool released from glycoproteins of the NORF yeast cells to predict the N-glycan uniformity. As this does not always accurately predict the N-glycan end product, we describe in this chapter a detailed protocol for a non-affinity-based high-throughput purification of an endogenous glycoprotein. This protein of interest has been introduced during the early stages of glycoengineering process and its N-glycan profile is utilized as a tool for glycoengineering screening.
Authors:
Sujatha Gomathinayagam; Erik Hoyt; Alissa M Thompson; Eric Brown; Khanita Karaveg; Stephen R Hamilton; Huijuan Li
Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Methods in molecular biology (Clifton, N.J.)     Volume:  899     ISSN:  1940-6029     ISO Abbreviation:  Methods Mol. Biol.     Publication Date:  2012  
Date Detail:
Created Date:  2012-06-27     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9214969     Medline TA:  Methods Mol Biol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  315-23     Citation Subset:  IM    
Affiliation:
GlycoFi Inc., A wholly owned subsidiary of Merck & Co Inc, Lebanon, NH, USA, sujatha_gomathinayagam@merck.com.
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