Design and Construction of His-tagged Protein Purification Workflow in Aquarium

Thesis (Master's)--University of Washington, 2019Protein purification is a long process starting from cloning, protein induction, extraction, isolation to protein verification. Carrying out this experiment can be very time-consuming and pricey, especially when some high-end instruments are required. Proper laboratory experiences and background knowledge of protein properties are also crucial for people who are conducting protein purification. As a result, the aforementioned considerations lead to the motivation of designing and building a robust protein purification workflow in Aquarium. The laboratory operating system, Aquarium, will compute required information such as protein concentration, sample amount, and buffer volume to yield step-by-step instructions for laboratory technicians for generating reproducible outcomes. The protein purification using poly-histidine-tag (His-tag) is implemented to provide an inexpensive purifying method. With Aquarium, the protocols are represented as executable code with negative controls. Moreover, the system automates multiple purifications in batches, which not only significantly saves time and budget, but also elevates the success rate. The success rate is verified by 10 identical purifications which are performed in 2 batches; each batch includes 5 purifications in parallel. This demonstrates that this workflow is capable of processing multiple batches of purification. The results of the test run also demonstrate this workflow is robust. The isopropyl-β-D-thio-galacto-pyranoside (IPTG) induced samples have a significant enrichment on the protein expression by comparing with uninduced samples. The high purity of purified proteins is verified by gel images of SDS-PAGE analysis as well as abundant protein yield is determined by NanoDrop. This workflow in Aquarium provides a robust, straightforward, economic and highly applicable tool to perform protein purification for further biomedical research