Fig. 4: CFE and AlphaLISA can be combined to prototype in vitro glycosylation reactions. | Nature Communications

Fig. 4: CFE and AlphaLISA can be combined to prototype in vitro glycosylation reactions.

From: Characterizing and engineering post-translational modifications with high-throughput cell-free expression

Fig. 4

a Schematic of the cell-free workflow. Nanodisc supplemented CFE reactions were first used to express CjPglB variants and then mixed with an acceptor protein containing a 6xHis tag and sequon and crude membrane fraction enriched with a bacterial glycan of interest. Samples were then analyzed using Protein A AlphaLISA donor beads and anti-6xHis AlphaLISA acceptor beads. b An IVG reaction using CjPglB, crude membrane fraction enriched with CPS from S. pneumoniae serotype 4, and sfGFP with a 6xHis tag and either (b) a DQNAT sequon or (c) an AQNAT sequon was serially diluted, mixed with varying concentrations of S. pneumoniae CPS4 antiserum, and analyzed via AlphaLISA. All data are representative of three independent experiments (n = 3). RLU relative luminescence units. Source data are provided in the Source Data 1 file.

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