Site-Specific Synthesis of Cysteine-Bridged Glycoproteins via Expressed Protein Glycoligation

  • Bioconjug Chem. 2020 Oct 21;31(10):2362-2366. doi: 10.1021/acs.bioconjchem.0c00437.
Nicholas Holloran  1 ,  Daniel Collins  1 ,  Upendra Rathnayake  1 ,  Bixia Zhang  1 ,  Minseob Koh  2 ,  ChulHee Kang  1 ,  Philip Garner  1
Affiliations
  • 1. Department of Chemistry, Washington State University, Pullman, Washington 99164-4630, United States.
  • 2. Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
PMID: 32931248 DOI: 10.1021/acs.bioconjchem.0c00437
Abstract

Site-specific glycosylation of a functional recombinant protein thioester is reported. The thioester functionalized protein sfGFP-Y151ThioD, prepared by genetic code expansion, underwent native chemical ligation with the cysteine-conjugated glycans H-Cys-NH-GlcNAc and H-Cys-NH-(GlcNAc)2(Man)3 to give the corresponding cysteine-bridged glycoproteins. The intact glycoproteins, which retained their fluorescence, were characterized by top-down mass spectrometry and gel electrophoresis. The bridging cysteine provided a convenient handle for affinity chromatography purification of the glycoproteins via a removable biotin tag. Given the influence that specific glycoforms can have on a protein's function, the ability to attach a homogeneous glycan to an intact protein in a functional group controlled yet sequon-independent manner could find widespread application. These preliminary results set the stage for development of the expressed protein glycoligation (EPG) concept.

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