Automated High-throughput Protein Purification with PhyTip® (PhyNexus) at Micro-scale

Abstract

High-throughput protein expression and purification have been successful tools to accelerate the finding of new targets in structural and functional studies in our group. We have routinely cloned and expressed varied constructs of a target protein simultaneously in different expression systems to rapidly identify the forms with the highest expression levels, stability and/or potency. Thus, an automated high-throughput micro-purification method was developed to process such numerous culture samples. The current methodology is based on robotic pipet tips - PhyTip® (PhyNexus) - prepacked with different types of chromatography resins (affinity, ionexchange, desalting etc.). The PhyTips are programmed to be transferred by a robot from protein samples to different purification buffers. Within a day, many plates of protein samples can be purified and analyzed by SDSPAGE, thus decisions can be made quickly on constructs to scale up. We show here how the Biomek® FX robot and PhyTips are set up; how we routinely use this technology to purify tagged proteins from different cell cultures to check for expression levels and stability ; and how quickly we can determine the forms and buffer conditions for scaling up.