title: 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 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.
authors.affiliation: Departments of Chemistry, Molecular Biology and Biochemistry, University of California, Irvine, CA, 92697-2025, USA.|Centre for NanoScale Science and Technology, Flinders University, Bedford Park, Adelaide, 5001, Australia.
authors.forename: Joshua
authors.initials: J
authors.lastname: Britton
authors.affiliation: Departments of Chemistry, Molecular Biology and Biochemistry, University of California, Irvine, CA, 92697-2025, USA.
authors.forename: Rebekah P
authors.initials: RP
authors.lastname: Dyer
authors.affiliation: Departments of Chemistry, Molecular Biology and Biochemistry, University of California, Irvine, CA, 92697-2025, USA.
authors.forename: Sudipta
authors.initials: S
authors.lastname: Majumdar
authors.affiliation: Centre for NanoScale Science and Technology, Flinders University, Bedford Park, Adelaide, 5001, Australia.
authors.forename: Colin L
authors.initials: CL
authors.lastname: Raston
authors.affiliation: Departments of Chemistry, Molecular Biology and Biochemistry, University of California, Irvine, CA, 92697-2025, USA.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Weiss
mesh_terms: D055162:Biocatalysis; D002846:Chromatography, Affinity / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification; D055767:Immobilized Proteins / Q000737:chemistry / Q000302:isolation & purification; D007535:Isomerases / Q000737:chemistry / Q000302:isolation & purification; D008164:Luminescent Proteins / Q000737:chemistry / Q000302:isolation & purification; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D013997:Time Factors; D014026:Nicotiana / Q000201:enzymology; D000097573:Red Fluorescent Protein
chemical_list: D029968:Escherichia coli Proteins; D055767:Immobilized Proteins; D007535:Isomerases; D008164:Luminescent Proteins; D008670:Metals; D011506:Proteins; D000097573:Red Fluorescent Protein; C060050:aristolochene synthase
keywords: biocatalysis; continuous flow; multi-step transformations; protein purification; thin films