Ten-Minute Protein Purification and Surface Tethering for Continuous-Flow Biocatalysis.

ABSTRACT: Nature applies enzymatic assembly lines to synthesize bioactive compounds. Inspired by such capabilities, we have developed a facile method for spatially segregating attached enzymes in a continuous-flow, vortex fluidic device (VFD). Fused His_n -tags at the protein termini allow rapid bioconjugation and consequent purification through complexation with immobilized metal affinity chromatography (IMAC) resin. Six proteins were purified from complex cell lysates to average homogeneities of 76 %. The most challenging to purify, tobacco epi-aristolochene synthase, was purified in only ten minutes from cell lysate to near homogeneity (>90 %). Furthermore, this "reaction-ready" system demonstrated excellent stability during five days of continuous-flow processing. Towards multi-step tr

SUBMITTER: Britton J 

PROVIDER: S-EPMC5480406 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature