Fig. 4
Mixing of CFGpS extracts enables rapid prototyping of different OST enzymes. a Western blot analysis of CFGpS reactions performed using lysate mixing strategy whereby CjLLO lysate derived from CLM24 cells carrying pMW07-pglΔB was mixed with CjPglB lysate derived from CLM24 cells carrying pSF-CjPglB, and the resulting CFGpS mixture was primed with plasmid DNA encoding either scFv13-R4DQNAT or sfGFP217-DQNAT. b Western blot analysis of CFGpS reactions performed using CjLLO lysate mixed with extract derived from CLM24 cells carrying a pSF plasmid encoding one of the following OSTs: CjPglB, CcPglB, DdPglB, DgPglB, or DvPglB. Mixed lysates were primed with plasmid DNA encoding either sfGFP217-DQNAT (D) or sfGFP217-AQNAT (A). Blots were probed with anti-His antibody to detect the acceptor proteins (top panels) and hR6 serum against the C. jejuni glycan (bottom panels). Arrows denote aglycosylated (g0) and singly glycosylated (g1) forms of the acceptor proteins. Molecular weight (MW) markers are indicated at left. Results are representative of at least three biological replicates
