A Novel Toolbox for Rapid, On Demand, In Vitro Glycoprotein Synthesis
Abstract
Protein therapeutics offer a significant opportunity to prophylactically protect against and to treat individuals following exposure to chemical and biological threat agents. For example, scavengers of fluorophosphonates based on acetylcholine esterases have emerged as important countermeasures to nerve agents. The use of defined glycoforms of the protein—where proteins are modified with sugars at defined positions—can be critical to optimizing the activities of therapeutic agents. Yet, the fundamental knowledge base to understand which glycoforms would be most effective is still lacking, as are methods that can prepare the glycoforms cost-effectively. This funded project is addressing this knowledge gap by developing technologies that can identify enzymes that are used to prepare the desired therapeutics. These tools include; (i) a cutting-edge cell-free expression system for rapidly synthesizing proteins; (ii) newly established methods that use bacterial approaches to control the attachment of sugars to proteins; and (iii) an extremely rapid screening tool that can optimize the preparation of a desired therapeutic. The project has the goals of preparing thousands of glycosylation enzymes and rapidly testing them to identify combinations of enzymes that selectively and efficiently convert an unmodified protein into a desired glycoform. This work will recapitulate glycan modulation using in vitro approaches and will enable the efficient preparation of human-like patterns of glycosylation that offer unprecedented control over immunogenicity, efficacy and stability of proteins. Key progress during the first year of the project includes the following. We have established a high yielding (capable of preparing greater than 1 gram of protein for each liter of culture) cell-free protein synthesis platform suitable for our glycosylation studies. We have also identified, cloned, and expressed multiple, active oligosaccharyltransferases (OSTs) and glycosyltransferases (GTs). For example, we expressed several soluble single-subunit OSTs, including two eukaryotic OSTs, through the use of cell-free protein synthesis. This progress is significant because the proteins are normally found in the cellular membrane, making them difficult to isolate in active form. We acquired the genes for an additional 100 OSTs and GTs and expect these enzymes to be useful in preparing the therapeutics outlined in the proposal. We have also developed a rapid test for the enzyme activities. This method, based on the SAMDI technology, uses plates having thousands of distinct reactions and further uses a mass spectrometry technique to rapidly read the result of each test. It is possible to perform more than 20,000 test in a single day and this work has already identified enzymes that have the desired activities.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jun 29, 2016
- Source ID
- HDTRA11510052
Entities
People
- Milan Mrksich
Organizations
- Defense Threat Reduction Agency
- Northwestern University