Capillary Electrophoresis as a High Throughput Screening Tool for Protein Protein Interactions.

Abstract

Protein Protein Pnteractions (PPIs) are key players in all aspects of cellular functions. In depth understanding of PPIs and discovery of PPI targeted therapeutics requires facile and user-friendly PPI analysis. Capillary Electrophoresis (CE) is a valuable tool to study affinity bindings including PPIs. Fast separation speed and nanoliter sample consumption suggest great potential for CE based High Throughput Screening (HTS). To explore CE as a PPI analysis and HTS tool, we have developed a CE based assay for Heat Shock Protein70 (Hsp70) and its co-chaperone Bcl2-Associated Anathogene 3 (Bag3). An Affinity Probe CE (APCE) method was developed for full length Hsp70 and Bag3 extracted from cell culture. In this assay, Hsp70 was labeled with Alexafluor® 488 and used as the affinity probe. With a surface modified capillary, the binding is detected as Hsp70 and complex peaks resolved and detected using Laser Induced Fluorescence (LIF). On a commercial instrument, the CE assay takes 6.5 min per run which allows a sample throughput of 220 tests per day. In a pilot screening, ~3300 small molecule library compounds were tested for novel inhibitor discovery. By comparing CE assay’s performance to an existing high throughput screening platform Flow Cytometry Protein Binding Assay (FCPIA), CE showed much better specificity as evidenced by lower hitting rate and higher confirmation rates in confirmatory tests. Fully realizing the low sample consumption benefit of CE based HTS requires low volume sample manipulation capability. Segmented flow microfluidics is an emerging technique that allows nanoliter to picoliter volume samples to be manipulated and coupled to CE analysis. In the second half of this thesis, unit operations of segmented flow microfluidics, namely splitting and reagent addition were developed to fulfill the need to aliquot and add reagent to nanoliter segmented flow samples. Based on these results, future work would be to integrate the CE separation with these sample manipulation techniques towards building a novel workflow for CE based HTS.