Work

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

Year: 2017

Type: article

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.

Source: Angewandte Chemie International Edition

DOI: 10.1002/anie.201610821

Authors: Joshua Britton, Rebekah P. Dyer, Sudipta Majumdar, Colin L. Raston, Gregory A. Weiss

Institutions: Flinders University, University of California, Irvine

Language: English

Topic: Enzyme Catalysis and Immobilization

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green

Funders: National Cancer Institute, National Institute of General Medical Sciences

Awards: Membrane Protein Co- Crystallization with Highly Crystalline and Soluble Proteins, A Training Program for Interdisciplinary Cancer Research