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Ten‐Minute Protein Purification and Surface Tethering for Continuous‐Flow Biocatalysis
Author(s) -
Britton Joshua,
Dyer Rebekah P.,
Majumdar Sudipta,
Raston Colin L.,
Weiss Gregory A.
Publication year - 2017
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201610821
Subject(s) - bioconjugation , flow chemistry , biocatalysis , lysis , chemistry , continuous flow , enzyme , immobilized enzyme , chromatography , affinity chromatography , fluidics , catalysis , combinatorial chemistry , nanotechnology , biochemistry , materials science , engineering , aerospace engineering , ionic liquid , physics , mechanics
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 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.