The OPPF-UK Pipeline for Structural Proteomics of Glycoproteins

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Technology Networks

The OPPF-UK has established a pipeline for the production of recombinant glycoproteins for structural proteomics. This high-throughput pipeline will be presented and includes all stages from construct design; through cloning and expression of protein in mammalian cells; and to protein purification, quality control and crystallization. The automated expression screening of glycoproteins by transient transfection in HEK 293T cells will be highlighted. This system allows for rapid, parallel assessment of expression level for up to 96 constructs. Technologies for both large scale transient expression and the production of protein via stable cell lines will be discussed along with examples showing the advantages of each system. To aid crystallization, glycans are modified by the use of HEK 293S cells or by growth in the presence of kifunensine. These methods give high-mannose glycans which are homogeneous and amenable to endoglycosidase H treatment. A number of case studies from different projects will be presented to illustrate use of the OPPF-UK high-throughput pipeline.