GlycoSCORES technique accelerates protein therapy research

A Northwestern-led synthetic biology research team has combined technologies to develop a new biotech technique that promises to accelerate research into protein therapies that could one day become the next defence against antibiotic-resistant super-germs or the next new drug.

Milan Mrksich, the Henry Wade Rogers Professor of Biomedical Engineering, Chemistry, and Cell and Molecular Biology, and colleague Michael Jewett, the Charles Deering McCormick Professor of Teaching Excellence and Associate Professor of Chemical and Biological Engineering, decided to compare notes and combine forces.

The pair wondered what they could accomplish if they combined the Mrksich lab’s mass spectrometry technology with the Jewett lab’s expertise in glycosylation and rapidly making proteins.

Glycosylation, which is the attachment of sugars to proteins, plays a critical role in how proteins form and work in cells and how cells interact with other cells. It is also important in the study of disease and biotechnologies.

Their ideas began to crystallise when they looped in glycosylation engineering expertise of close collaborator Matt DeLisa, the William L. Lewis Professor of Engineering at the Robert Fredrick Smith School of Chemical and Biomolecular Engineering at Cornell University.

Together they developed a new platform for characterising and optimising sequences for making glycoproteins using cell-free protein synthesis and mass spectrometry.

The new technique promises to vastly speed up the time needed to test compounds for potential new drugs. As recent as a few decades ago, drugs were based on natural products that were tediously isolated and characterised from plants and other natural sources.

But once chemists learned to make libraries of large numbers of molecules – which today number in the millions – and once engineering brought laboratory automation forward as a tool, scientists and engineers were able to rapidly test millions of compounds within a few weeks to identify good starting points for drug development.

Still, Prof Mrksich explained, in synthetic biology, the cycle time to test each enzyme-substrate interaction can take weeks or months.

“We have radically accelerated the process,” Prof Mrksich said. “Where researchers today can evaluate a couple of hundred potential glycosylation tags in a given period, we’ve brought together two high-throughput technologies that allow us to evaluate several thousand in that same time frame.” These tags are important because glycosylation is present in 70 percent of protein therapeutics already approved or in preclinical evaluation.

The process works by combining three techniques:

• Cell-free protein synthesis
• Protein glycosylation
• SAMDI (self-assembled monolayers for matrix-assisted desorption/ionisation) mass spectrometry.

Combined with the glycoengineering knowledge from Dr DeLisa’s lab, the combined technique analyses glycosylation quickly and effectively.

“We developed peptide arrays where we have one plate about the size of your hand that has approximately 1,500 circular regions on it,” Prof Mrksich said. “Each of those regions has attached to it a different peptide tag, and we can apply the enzyme solution evenly across the full array and each of the peptide tags can then be glycosylated.”

After the plate is rinsed, the entire array can be analysed by mass spectrometry, which quantifies the amount of glycosylation of each peptide.

“In a day, we can evaluate thousands of distinct peptide tags to identify the optimal ones for glycosylation that we then move forward with,” Mrksich said.

The result is not only much faster but also delivers much more detailed data. “Our method allows us to not just pick the winners, which we commonly look for in scientific experiments, but the failures too,” Jewett said.

The team dubbed the process GlycoSCORES, or glycosylation sequence characterisation and optimisation by rapid expression and screening.

Dr DeLisa said he was excited to use the new technology to address a number of open questions regarding how different glycosylation enzymes work.

“This technique allows us to ask much more precise and scientific questions in this area than would have been previously possible,” he said. “The new knowledge that is derived could really be game-changing in terms of our ability to engineer glycoproteins with desirable traits.”

The resulting advance is described in “Design of glycosylation sites by rapid synthesis and analysis of glycosyltransferases,” published in the journal Nature Chemical Biology.