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Ten-Minute Protein Purification and Surface Tethering for Continuous-Flow Biocatalysis
Joshua Britton1,2, Rebekah P Dyer1, Sudipta Majumdar1
1Departments of Chemistry, Molecular Biology and Biochemistry, University of California, Irvine, CA, 92697-2025, USA.
Angewandte Chemie (International Ed. in English)
|January 31, 2017
Summary
We developed a new method to spatially segregate enzymes in a continuous-flow vortex fluidic device (VFD). This system enables efficient purification and ordered enzyme arrangement for continuous-flow biosynthesis.
Area of Science:
- Biotechnology
- Chemical Engineering
- Synthetic Biology
Background:
- Nature utilizes enzymatic assembly lines for complex bioactive compound synthesis.
- Mimicking natural enzymatic processes is crucial for advancing synthetic biology and biocatalysis.
Purpose of the Study:
- To develop a facile method for spatially segregating enzymes in a continuous-flow vortex fluidic device (VFD).
- To enable efficient purification and ordered arrangement of enzymes for multi-step biosynthesis in flow.
Main Methods:
- Enzymes were engineered with fused His-tags for rapid bioconjugation.
- Immobilized metal affinity chromatography (IMAC) resin was used for enzyme purification.
- A vortex fluidic device (VFD) was employed for spatial enzyme segregation and continuous-flow processing.
Main Results:
- Six proteins were purified from complex cell lysates with an average homogeneity of 76%.
- Tobacco epi-aristolochene synthase was purified to >90% homogeneity in just 10 minutes.
- The system demonstrated excellent stability over five days of continuous-flow processing.
Conclusions:
- The developed VFD system provides an efficient method for enzyme purification and spatial organization.
- This approach facilitates multi-step enzymatic transformations in a continuous-flow format.
- Enzyme zoning in VFDs opens new avenues for continuous-flow biosynthesis and biocatalysis.
