A fully automated robotic system for purification of polyhistidine‐tagged proteins

Abstract

A protein isolation method that results in rapid and high yield proteins is essential for protein sample preparation in proteomics. A magnetic bead-based automated method to purify both soluble and insoluble 6x histidine-tag (His-tag) proteins from cell lysates has been developed employing PSS Magtration® technology. Magtration® is a PSS patented technique that traps magnetic particles against the sidewall of a disposable pipette tip, resulting in superior washing and recovery compared to other magnetic particle-separation systems. We have extended this technology from immunoassay and nucleic acid purification to His-tag protein purification using Magtration® 12GC robotic system with commercially available Ni2+ and Co2+ magnetic beads. Unlike other automated protein purification systems, Magtration®12GC allows for high volumes (up to 800 μl) of initial sample, and can run up to 12 samples at once. Experimental conditions including buffer compositions, buffer pH and various His-tag protein binding magnetic beads have been studied and optimized for the robotic purification on the Magtration® system. A two-step elution protocol has been developed for effective removal of nonspecific proteins resulting in highly pure His-tag target proteins. The yield and purity of the His-tag protein from the automated system are more successful in terms of efficiency and reproducibility compared to manual protocols due to more thorough washing, complete separation between the magnetic beads and supernatant, and consistent robotic operation.