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

被引：49

作者：

Britton, Joshua ^{[1
,2
,3
,4
]}

Dyer, Rebekah P. ^{[1
,2
,3
]}

Majumdar, Sudipta ^{[1
,2
,3
]}

Raston, Colin L. ^{[4
]}

Weiss, Gregory A. ^{[1
,2
,3
]}

机构：

[1] Univ Calif Irvine, Dept Chem, Irvine, CA 92697 USA

[2] Univ Calif Irvine, Dept Mol Biol, Irvine, CA 92697 USA

[3] Univ Calif Irvine, Dept Biochem, Irvine, CA 92697 USA

[4] Flinders Univ S Australia, Ctr NanoScale Sci & Technol, Bedford Pk, SA 5001, Australia

来源：

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION | 2017年 / 56卷 / 09期

基金：

澳大利亚研究理事会;

关键词：

biocatalysis; continuous flow; multi-step transformations; protein purification; thin films; PRODUCT ASSEMBLY LINES; MULTISTEP SYNTHESIS; VORTEX FLUIDICS; IMMOBILIZED ENZYMES; POLYKETIDE; MICROREACTOR; CHEMISTRY; DESIGN; SYSTEM;

D O I：

10.1002/anie.201610821

中图分类号：

O6 [化学];

学科分类号：

0703 ;

摘要：

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.

引用

页码：2296 / 2301

页数：6

共 44 条

[1] On-demand continuous-flow production of pharmaceuticals in a compact, reconfigurable system [J].

Adamo, Andrea ;

Beingessner, Rachel L. ;

Behnam, Mohsen ;

Chen, Jie ;

Jamison, Timothy F. ;

Jensen, Klavs F. ;

Monbaliu, Jean-Christophe M. ;

Myerson, Allan S. ;

Revalor, Eve M. ;

Snead, David R. ;

Stelzer, Torsten ;

Weeranoppanant, Nopphon ;

Wong, Shin Yee ;

Zhang, Ping .

SCIENCE, 2016, 352 (6281) :61-67

[2]

[Anonymous], 2016, Angew. Chem

[3]

[Anonymous], 2012, ANGEW CHEM

[4]

Bogdan A. R., 2009, ANGEW CHEM, V121, P8699

[5] The Continuous-Flow Synthesis of Ibuprofen [J].

Bogdan, Andrew R. ;

Poe, Sarah L. ;

Kubis, Daniel C. ;

Broadwater, Steven J. ;

McQuade, D. Tyler .

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 2009, 48 (45) :8547-8550

[6] Engineering the third wave of biocatalysis [J].

Bornscheuer, U. T. ;

Huisman, G. W. ;

Kazlauskas, R. J. ;

Lutz, S. ;

Moore, J. C. ;

Robins, K. .

NATURE, 2012, 485 (7397) :185-194

[7] Accelerating Enzymatic Catalysis Using Vortex Fluidics [J].

Britton, Joshua ;

Meneghini, Luz M. ;

Raston, Colin L. ;

Weiss, Gregory A. .

ANGEWANDTE CHEMIE-INTERNATIONAL EDITION, 2016, 55 (38) :11387-11391

[8] Rapid protein immobilization for thin film continuous flow biocatalysis [J].

Britton, Joshua ;

Raston, Colin L. ;

Weiss, Gregory A. .

CHEMICAL COMMUNICATIONS, 2016, 52 (66) :10159-10162

[9] Harnessing Thin-Film Continuous-Flow Assembly Lines [J].

Britton, Joshua ;

Castle, Jared W. ;

Weiss, Gregory A. ;

Raston, Colin L. .

CHEMISTRY-A EUROPEAN JOURNAL, 2016, 22 (31) :10773-10776

[10] The synthesis of di-carboxylate esters using continuous flow vortex fluidics [J].

Britton, Joshua ;

Dalziel, Stuart B. ;

Raston, Colin L. .

GREEN CHEMISTRY, 2016, 18 (07) :2193-2200

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