Selective Organic Precipitation/Extraction of Released N-Glycans Following Large-Scale Enzymatic Deglycosylation of Glycoproteins
✍ Scribed by Mary Frances Verostek; Catherine Lubowski; Robert B. Trimble
- Book ID
- 102561916
- Publisher
- Elsevier Science
- Year
- 2000
- Tongue
- English
- Weight
- 317 KB
- Volume
- 278
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A major difficulty with isolating enzymatically or chemically released oligosaccharides from large-scale glycoprotein deglycosylation reactions is the timeconsuming chromatography, desalting, and concentration steps required to prepare a glycan fraction of manageable proportions. To overcome these time and preparative chromatography equipment requirements, we have developed a rapid organic solvent precipitation/extraction procedure that allows sequential isolation of endo--N-acetylglucosaminidase H (EC 3.2.1.96)-released high-mannose and hybrid, peptide-N 4 -(N-acetyl--glucosaminyl) Asn amidase (EC 3.5.1.52)-released complex, and -eliminated O-linked glycans without the need for intermediate chromatography, desalting, or concentration steps. The method involves precipitation of protein and released glycans at ؊20°C in 80% acetone and extraction of the glycans from the pellet with 60% aqueous methanol after each deglycosylation step. Three pools of essentially saltand detergent-free oligosaccharides (high-mannose/ hybrid, complex, and O-linked) can be isolated in a high yield in 4 days with this protocol, which has been extensively tested using bovine RNase B, human bile salt-stimulated lipase expressed in Pichia pastoris, hen ovalbumin, bovine fetuin, bovine thyroglobulin, and several invertase preparations from wild-type and mutant yeast strains.