Expression of appropriately glycosylated proteins in mammalian cells can be a challenging and expensive task. A less expensive alternative is the use of genetically manipulated plant-based systems. But can plant cells, with their different glycosylation machinery, decorate mammalian proteins properly?
A team of researchers at the University of California, Davis, are tackling this complex problem. Led by Raymond Rodriguez, Ph.D., a professor of molecular and cellular biology, and Karen A. McDonald, Ph.D., a professor of chemical engineering and materials science, the Davis team is carrying out an interdisciplinary program—combining plant science, chemistry, chemical engineering, and molecular biology—to achieve its goal of in vitro, post-production sialylation of an important human enzyme.
“We performed collaborative studies in which plant cells are used to express recombinant human butyrylcholinesterase (rhBChE), a serum-based bioscavenger for neurotoxic organophosphates (OP) like the deadly compound sarin,” said Dr. McDonald. “Current therapies are designed to elevate serum levels of BChE, but a single dose can cost as much as $10,000. We performed studies to express rhBChE in plants utilizing a novel means to create appropriate modifications in post-production.”
The Davis team utilized viral amplicon-based gene expression systems that compared rhBChE production via Tobacco mosaic virus versus Cucumber mosaic virus in Nicotiana benthamiana, a relative of tobacco and a commonly used model organism in plant research.
According to Dr. Rodriguez, “Development of rhBChE is a pressing national security issue. The U.S. government is seeking economical ways to mass produce this enzyme. However, BChE is a real challenge as it is a tetrameric protein with each monomer housing nine potential N-glycosylation sites. We targeted the protein to different subcellular compartments and found important differences in how the protein was glycosylated.”
Another novel aspect of the studies dealt with in vitro sialylation, the final “polishing” step for N-glycans. “Sialylation of glycoproteins is an end-stage modification that is critical for many processes such as maintaining stability, protein half-life, and immunogenic properties,” commented Xi Chen, Ph.D., professor of chemistry. “Since plants are incapable of sialylating glycoproteins, we employed post-production multistep enzymatic reactions to systematically add sialic acid to the termini of the N-glycans.”
Dr. McDonald noted that although the Davis team is in the early stages of bioengineering plant-based systems, the results are already encouraging. “We will continue to optimize these systems and also incorporate computational modeling of glycoproteins. Given that more than 30% of all commercial biopharmaceuticals are glycoproteins, our results suggest plant-based systems are viable alternatives to standard mammalian and insect expression systems.”