Fig. 2.

In vitro synthesis and assembly of one- and two-enzyme glycosylation pathways. a Protein name, species, previously characterized activity and optimized soluble CFPS yields for Im7-6 target protein, ApNGT, and GTs selected for glycan elaboration. References for previously characterized activities in Supplementary Table 4. CFPS yields indicate mean and standard deviation (s.d.) from n = 3 CFPS reactions quantified by [¹⁴C]-leucine incorporation. Full CFPS expression data in Supplementary Table 2 and Supplementary Figs. 1–2. b Symbol key and successful pathways for N-linked glucose installation on Im7-6 by ApNGT and elaboration by selected GTs. Glycan structures in this article use Symbol Nomenclature for Glycans (SNFG) and Oxford System conventions for linkages. Sialic acid refers to N-acetylneuraminic acid. c Deconvoluted mass spectrometry spectra from Im7-6 protein purified from IVG reactions assembled from CFPS reaction products with and without 0.4 µM ApNGT as well as 2.5 mM UDP-Glc. Full conversion to N-linked glucose was observed after 24 h at 30 °C. d Intact deconvoluted MS spectra from Im7 protein purified from IVG reactions containing 10 µM Im7-6, 0.4 µM ApNGT, and 7.8 µM NmLgtB, 13.9 µM NgLgtB, 3.1 µM BfGalNAcT, or 9.4 µM Apα1-6. IVG reactions were supplemented with 2.5 mM UDP-Glc as well as 2.5 mM UDP-Gal or 5 mM UDP-GalNAc as appropriate for 24 h at 30 °C. Observed mass shifts and MS/MS fragmentation spectra (Supplementary Fig. 3) are consistent with efficient modification of N-linked glucose with β1-4Gal, β1-4Gal, β1-3GalNAc, or α1-6 dextran polymer. Theoretical protein masses found in Supplementary Table 3. Hpβ4GalT, Btβ4GalT1, and SpWchJ + K did not modify the N-linked glucose installed by ApNGT (Supplementary Fig. 4). All spectra were acquired from full elution peak areas of all detected glycosylated and aglycosylated Im7-6 species and are representative of n = 3 independent IVGs. Spectra from m/z 100–2000 were deconvoluted into 11,000–14,000 Da using Bruker Compass Data Analysis maximum entropy method. Source data is available in the Source Data file