Researcher(s)
- Zachary Dixon, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Mark Blenner, Chemical and Biomolecular Engineering, University of Delaware
Abstract
Chinese hamster ovary (CHO) cells are commonly used for industrial monoclonal antibody (mAb) and bispecific antibody (bsAb) production. For production to match the growing market for antibodies, new targets for cell engineering must continually be identified. Targets identified via proteomic-based approaches are well documented; however, studies sparsely focus attention on the classical secretory pathway (CSP), which has been identified as a bottleneck during recombinant protein production. One approach that can be used is a spatial-proteomic approach involving proximity-labeling proteins (PLPs) can be used to identify CSP-residing targets directly involved in mAb and bsAb folding and processing. In preparation for identifying these targets, this work aimed to establish PLP-producing cell lines, demonstrate PLP functionality, and optimize purification methods for molecules modified by PLPs. Plasmids encoding the PLPs APEX2 and ultraID were cloned using Golden Gate (GG) and Gibson assembly reactions. GG-assembled plasmids were stably integrated into NIST CHO cells for future PLP analysis. Gibson-assembled plasmids were transformed into the BL21 E. coli strain. PLP expression in BL21 was confirmed via protein gel and activity was assessed via Western blot. Interestingly, results confirm ultraID labeling activity but refute APEX2 labeling activity, suggesting non-optimal conditions for APEX2. Further, biotinylated protein purification was performed on cell lysates using Strep-Tactin® MagBeads. Bradford assay results indicate that this method failed to capture a measurable amount of protein and is therefore unsuited for biotinylated protein purification. Future work will aim to demonstrate PLP expressions and activity in CHO cells, improve APEX2 labeling conditions, and identify an alternative method for biotinylated protein purification.