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

Joshua Britton, Rebekah Dyer, Sudipta Majumdar, Colin Raston, Gregory Weiss

    Research output: Contribution to journalArticle

    22 Citations (Scopus)

    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 Hisn-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 transformations in continuous flow, proteins were arrayed as ordered zones on the reactor surface allowing segregation of catalysts. Ordering enzymes into zones opens up new opportunities for continuous-flow biosynthesis.

    Original languageEnglish
    Pages (from-to)2296-2301
    Number of pages6
    JournalAngewandte Chemie-International Edition
    Volume56
    Issue number9
    DOIs
    Publication statusPublished - 2017

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