Developing an in Vitro Platform for the Production of Structurally Homogeneous Glycoproteins Using Cell-Free Protein Synthesis and Samdi Mass Spectrometry

Glycosylation, the attachment of complex oligosaccharides (glycans) to proteins, is the most abundant polypeptide chain modification in nature and plays a pivotal role in protein folding, quality control, sorting, degradation, secretion and activity. Glycoproteins make up 70% of approved or preclinical protein therapeutics and a predicted two-thirds of human proteins. Changes in glycosylation are often a hallmark of cancer, inflammation, and Alzheimer’s disease states and have been shown to profoundly affect therapeutically relevant protein properties including pharmacokinetics, immunogenicity, and biological activity. In fact, intentional engineering of a protein’s glycosylation pattern has led to therapeutics with improved efficacy. However, the fundamental knowledge base and experimental techniques required to systematically assess which human glycoforms would be most effective at a given site are still lacking, as are scalable methods to site-specifically conjugate desired glycoforms to proteins. These knowledge and technology gaps stem from challenges in analysis of glycoforms and the complexity of glycan biosynthesis pathways. Glycan production is not template driven but is accomplished by the coordinated expression, regulation, and localization of glycosyltransferases (GTs) and oligosaccharyltransferases (OSTs) in various cellular compartments. The complex biosynthesis leads to a massive diversity of glycan structures. The complexity of this mixture has prohibited a systematic analysis of the effect of a particular glycan structure on glycoprotein function and has impeded efforts to engineer glycoprotein therapeutics with desired properties.

We will discuss our efforts to develop a cell-free expression platform where synthetic glycan biosynthesis pathways can be constructed and carefully controlled to yield structurally homogeneous glycoproteins. The open reaction environment of our in vitro glycosylation system allows us to apply a unit operations approach in which homogeneous glycans are installed on a target protein and elaborated in a step-wise fashion by rigorously controlling the reaction conditions, enzyme composition, and residence time in various compartments which mimic specific cellular organelles. In order to identify enzymes with desired specificities and kinetic profiles, we have expressed dozens of N-glycan modifying GT and OST enzymes from bacteria, human, mouse, and Chinese Hamster Ovary cell lines in E. coli based cell-free protein synthesis (CFPS) systems. By manipulating the redox environment and adding membrane mimics, we have achieved soluble titers of 150-700 μg/μL of these diverse enzymes that are often difficult to express in vivo. Unlike yeast or CHO systems, E. coli lysates lack native glycosylation machinery, providing a blank canvas for bottom-up glycoengineering.

In order to determine ideal enzyme and substrate combinations and reaction conditions for this in vitro platform, we are combining our CFPS system with a high-throughput, label-free screening tool known as SAMDI (Self-Assembled Monolayers for matrix-assisted Laser Desorption/Ionization) mass spectrometry. Here we report the synthesis and characterization of GTs and OSTs in crude lysates by screening their activity on immobilized glycosylation substrates.

By harnessing the versatility and throughput of cell-free systems, we hope that our glycoprotein synthesis platform 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.