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Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
Published on: January 16, 2012
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Orthogonal Surface Tags for Whole-Cell Biocatalysis
Theo Peschke1, Kersten S Rabe1, Christof M Niemeyer1
1Karlsruhe Institute of Technology (KIT), Institute for Biological Interfaces (IBG 1), Hermann-von-Helmholtz-Platz, 76344, Eggenstein-Leopoldshafen, Germany.
Angewandte Chemie (International Ed. in English)
|January 21, 2017
Summary
Engineered E. coli strains display orthogonal tags for surface immobilization and express functional proteins in the cytosol. This enables selective binding to substrates and efficient biocatalysis for specific chemical transformations.
Area of Science:
- Synthetic Biology
- Biotechnology
- Microbial Engineering
Background:
- Cell surface display is crucial for immobilizing microbial catalysts.
- Simultaneous intracellular protein expression is needed for biocatalytic functions.
- Current methods often lack orthogonality or efficiency.
Purpose of the Study:
- To engineer Escherichia coli (E. coli) strains for simultaneous cell surface display and intracellular protein expression.
- To develop orthogonal tagging systems for robust immobilization.
- To demonstrate the utility of these engineered strains in biocatalysis.
Main Methods:
- Utilized the Lpp-ompA outer membrane protein for cell surface display.
- Employed orthogonal tags: streptavidin-binding peptide, SpyTag/SpyCatcher, and HaloTag variants.
- Co-expressed heterologous functional proteins (fluorescent proteins, ketoreductase enzymes) in the cytosol.
- Validated immobilization using magnetic microbeads.
- Assessed biocatalytic activity using a prochiral nitrodiketone substrate.
Main Results:
- Successfully generated E. coli strains with surface-displayed orthogonal tags for selective immobilization.
- Demonstrated simultaneous cytosolic expression of functional fluorescent proteins and ketoreductase enzymes.
- Achieved high selectivity in immobilization onto complementary surfaces.
- Engineered strains exhibited high stereoselectivity in whole-cell biocatalysis of a nitrodiketone.
Conclusions:
- Developed a versatile platform for engineering E. coli with dual functionality: surface immobilization and intracellular biocatalysis.
- Orthogonal tagging systems provide precise control over cell attachment to substrates.
- These engineered microbes show significant potential for applications in biocatalysis and bioprocessing.
