Glycosylation Issues in Purification
The Oxford Protein Production Facility (OPPF) is a structural proteomics facility at Oxford University, funded by the Medical Research Council. The group’s work in dealing with glycosylation issues in protein purification will be reviewed by Joanne Nettleship, Ph.D., senior scientist, at the meeting.
“We’re involved in high-throughput technologies using 96-well plates to clone, express, and purify proteins for U.K. academics,” says Dr. Nettleship, summarizing the overall mission of the facility.
The lab’s small-scale expression protocols allow for fast, effective assessment of expression level from a large number of plasmids in parallel. The level of secreted and intracellular protein expression can be assessed within a week of cloning the gene of interest. “These diverse needs call on the use of human HEK293 cells, E. coli, and baculovirus expression systems.”
Glycosylation is necessary for property folding of many eukaryotic proteins, so these situations require the use of the HEK293 cell line. “For some of our more challenging proteins, we may look at the performance of more than one expression system,” she explains.
Dr. Nettleship favors transient expression, since in a high-throughput laboratory where rapid screening of many constructs is required and repeated expression experiments are not needed, the transient system is less time consuming.
Given that many investigators need homogeneous proteins in order to make crystals for x-ray crystallographic analysis, Dr. Nettleship and her colleagues have optimized approaches that ensure that glycosylation will be uniform through the application of kifunensine, the a-mannosidase I inhibitor. This leaves the proteins in a high mannose state, after which they can be treated with mannosidase to achieve homogeneity of the product.
Dr. Nettleship and her colleagues on the OPPF team set in motion and manage on an ongoing basis the high-throughput crystallization facility. “Using laboratory automation, we have established a protein-production pipeline capable of processing a thousand input sequences per year,” she concludes.