Protein Purification Handbook

  • Temming R
  • van Scherpenzeel M
  • Te Brinke E
 et al. 
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Abstract

An enrichment strategy was devised for azide derivatized macromolecules, based on strain-promoted alkyne-azide cycloaddition (SPAAC) and a cleavable linker. A ring-strained alkyne, bicyclo[6.1.0]non-4-yne (BCN), was covalently attached to agarose beads via a hydrazine-sensitive linker. Benchmark studies of the resulting 'azido-trap' beads were performed with a fluorogenic coumarin derivative, leading to efficient capture of the azidocoumarin with concomitant fluorescence staining of the beads via SPAAC. The versatility of the beads for specific protein enrichment was shown by an effective and highly specific capture-release strategy for enrichment of azido-containing Candida antarctica lipase B (CalB) from a mixture of proteins. This approach is suited for selective enrichment of (glyco)proteins after metabolic incorporation of azides for subsequent (glyco)proteomics studies.

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Authors

  • Rinske P Temming

  • Monique van Scherpenzeel

  • Esra Te Brinke

  • Sanne Schoffelen

  • Jolein Gloerich

  • Dirk J Lefeber

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