Journal: Journal of Biological Chemistry

Article Title: Enzymatic Basis for N-Glycan Sialylation

doi: 10.1074/jbc.m113.519041

Figure Lengend Snippet: FIGURE 2. Strategy for ST6GAL1 expression, purification, and tag/glycan cleavage. A diagrammatic representation of the coding region for the recombi- nant ST6GAL1 fusion protein expression product is shown at the top of the figure. The expression construct encoded a fusion protein containing an NH2- terminal signal sequence followed by an His8 tag, AviTag, superfolder GFP, TEV protease cleavage site, and the catalytic domain of ST6GAL1 (see “Experimental Procedures” for details). Expression of the recombinant product in HEK293S cells resulted in the secretion of the fusion protein into the culture medium (crude media, lower panel), and subsequent Ni-NTA purification yielded a highly enriched enzyme preparation (IMAC1 elution, lower panel). Cleavage of the enzyme with TEV protease and EndoF1 resulted in removal of tag sequences and glycans down to a single GlcNAc residue attached to the peptide backbone (TEV EndoF1, lower panel). Chromatography over Ni-NTA resulted in the elution of the ST6GAL1 catalytic domain and retention of the tag sequences, TEV protease, and EndoF1 on the column by virtue of their His tags (IMAC2 elution). The enzyme was further purified over Superdex-75, and peak fractions from the gel filtration column are shown in the lower panel. The final pool of purified ST6GAL1 (gel filtration pool) resulted in an 80% yield from the crude culture medium.

Article Snippet: Wild type suspension culture HEK293 cells (FreeStyle 293-F cells, Invitrogen) or mutant HEK293S GnTI cells (35) (ATCC catalog no. CRL-3022) were maintained at 0.5–3.0 106 cells/ml in a humidified CO2 platform shaker incubator at 37 °C.

Techniques: Expressing, Purification, Glycoproteomics, Construct, Sequencing, Recombinant, Residue, Chromatography, Filtration