High-throughput multimodal strong anion exchange purification and N-glycan characterization of endogenous glycoprotein expressed in glycoengineered Pichia pastoris.
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.
GlycoFi Inc., A wholly owned subsidiary of Merck & Co Inc, Lebanon, NH, USA. sujatha_gomathinayagam@merck.com
Sujatha
S
Gomathinayagam
Erik
E
Hoyt
Alissa M
AM
Thompson
Eric
E
Brown
Khanita
K
Karaveg
Stephen R
SR
Hamilton
Huijuan
H
Li
D000837:Anion Exchange Resins / Q000737:chemistry; D002852:Chromatography, Ion Exchange; D006023:Glycoproteins / Q000302:isolation & purification*; D006031:Glycosylation; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D010843:Pichia; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering
D000837:Anion Exchange Resins; D006023:Glycoproteins; D011134:Polysaccharides