Design of Protein Glycosylation Sites By Cell-Free Protein Synthesis and Mass Spectrometry of Self-Assembled Monolayers
- Conference: Synthetic Biology Engineering Evolution Design SEED
- Year: 2017
- Proceeding: 2017 Synthetic Biology: Engineering, Evolution & Design (SEED)
- Group: General Submissions
Tuesday, June 20, 2017 - 2:30pm-2:45pm
Here we describe an in vitro platform for high-throughput expression and characterization of glycosylation enzymes using E. coli Cell-Free Protein Synthesis (CFPS) and Self-Assembled Monolayers for Desorption Ionization Mass Spectrometry (SAMDI-MS). This workflow allowed us to produce >800 µg/mL of a cytoplasmic N-linked glycosyltransferase (NGT) in vitro and determine its peptide acceptor and sugar donor specificities at unprecedented depth and throughput with ~10,000 unique reactions conditions and ~3,000 unique peptide substrates. We used this dataset to develop a small, robust acceptor sequence motif (GlycTag) to direct the efficient installation of N-linked glycans onto the internal loops of heterologous protein substrates in vitro and in the cytoplasm of living E. coli. We found that SAMDI-MS observations at the peptide level accurately predicted modification trends in the context of whole proteins. We placed GlycTags in three structurally diverse target proteins, including the Fc region of a human antibody, and found that these sequences were modified at 3-5 fold greater efficiency compared to wild-type glycosylation sequence motifs. Notably, this N-linked glycosylation system does not require protein transport across cellular membranes or the use of membrane bound components, making it particularly attractive for the development of synthetic glycosylation systems.
This work provides a disruptive technique for glycosyltransferase characterization and provides a new set of tools towards emerging bacterial and in vitro glycosylation platforms. Ultimately, these glycoengineering efforts will enable a deeper understanding of glycan structure and function and help to bring about a new generation of rationally designed glycoprotein therapeutics, vaccines, and diagnostics.