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Covalent Functionalization of Bioengineered Polyhydroxyalkanoate Spheres Directed by Specific Protein-Protein
Jin Xiang Wong1,2, Majela Gonzalez-Miro3, Andrew J Sutherland-Smith1
1School of Fundamental Sciences, Massey University, Palmerston North, New Zealand.
Frontiers in Bioengineering and Biotechnology
|March 3, 2020
Summary
Engineered bacteria create functional polyhydroxyalkanoate (PHA) spheres for protein immobilization. This novel method uses SpyTag/SpyCatcher ligation for efficient, specific surface functionalization, enabling versatile applications.
Area of Science:
- Synthetic Biology
- Biotechnology
- Protein Engineering
Background:
- Bioengineered polyhydroxyalkanoate (PHA) spheres offer potential for protein immobilization.
- Existing methods often require purified proteins and lack efficiency in complex mixtures.
Purpose of the Study:
- To develop streamlined, specific methods for functionalizing PHA spheres with proteins.
- To demonstrate the versatility and robustness of protein ligation strategies for PHA sphere modification.
Main Methods:
- Engineered *Escherichia coli* to co-assemble PHA spheres displaying SpyCatcher and produce SpyTagged proteins *in vivo*.
- Utilized SpyTag/SpyCatcher mediated protein ligation for direct functionalization of PHA spheres from complex mixtures.
- Developed bimodular PHA spheres using alternative tag/catcher systems (SnoopTag/SnoopCatcher, SdyTag/SdyCatcher) for multifunctionalization.
Main Results:
- Achieved specific *in vivo* and *in vitro* ligation of target proteins to SpyCatcher-displaying PHA spheres, even from crude cell lysates.
- Demonstrated one-step simultaneous multifunctionalization of PHA spheres with two different proteins using bimodular systems.
- Confirmed orthogonality with non-target tags and robustness against freeze-thaw cycles.
Conclusions:
- Established a versatile toolbox for dynamic functionalization of PHA spheres using modular protein ligation.
- The developed approaches enable efficient protein immobilization for diverse biomedical and industrial applications.
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