Glycoengineering of human cell lines using zinc finger nuclease gene targeting: SimpleCells with homogeneous GalNAc O-glycosylation allow isolation of the O-glycoproteome by one-step lectin affinity chromatography

Methods Mol Biol. 2013;1022:387-402. doi: 10.1007/978-1-62703-465-4_29.

Abstract

Lectin affinity chromatography is a powerful technique for isolation of glycoproteins carrying a specific glycan structure of interest. However, the enormous diversity of glycans present on the cell surface, as well as on individual proteins, makes it difficult to isolate an entire glycoproteome with one or even a series of lectins. Here we present a technique to generate cell lines with homogenous truncated O-glycans using zinc finger nuclease gene targeting. Because of their simplified O-glycoproteome, the cells have been named SimpleCells. Glycoproteins from SimpleCells can be isolated in a single purification step by lectin chromatography performed on a long lectin column. This protocol describes Zinc finger nuclease gene targeting of human cells to simplify the glycoproteome, as well as lectin chromatography and isolation of glycopeptides from total cell lysates of SimpleCells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Culture Techniques / methods
  • Cell Engineering / methods*
  • Cell Line
  • Chromatography, Affinity / methods*
  • Endoribonucleases / genetics
  • Endoribonucleases / metabolism
  • Gene Targeting / methods
  • Glycoproteins / analysis
  • Glycoproteins / isolation & purification*
  • Glycoproteins / metabolism
  • Glycosylation
  • Humans
  • Immunohistochemistry / methods
  • Lectins / metabolism
  • Proteome / analysis
  • Proteome / isolation & purification*
  • Proteome / metabolism
  • Proteomics / methods
  • Zinc Fingers

Substances

  • Glycoproteins
  • Lectins
  • Proteome
  • Endoribonucleases