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Updated: Aug 20, 2025
Co-Translational Insertion of Membrane Proteins into Preformed Nanodiscs
Published on: November 19, 2020
Cell-Free Protein Synthesis of Particulate Methane Monooxygenase into Nanodiscs
Christopher W Koo1, Jasmine M Hershewe2, Michael C Jewett2,3
1Department of Molecular Biosciences and of Chemistry, Northwestern University, Evanston, Illinois60208, United States.
Abstract:
Particulate methane monooxygenase (pMMO) is a multi-subunit membrane metalloenzyme used by methanotrophic bacteria to convert methane to methanol. A major hurdle to studying pMMO is the lack of a recombinant expression system, precluding investigation of individual residues by mutagenesis and hampering a complete understanding of its mechanism. Here, we developed an Escherichia coli lysate-based cell-free protein synthesis (CFPS) system that can be used to express pMMO in vitro in the presence of nanodiscs. We used a SUMO fusion construct to generate the native PmoB subunit and showed that the SUMO protease (Ulp1) cleaves the protein in the reaction mixture. Using an affinity tag to isolate the complete pMMO complex, we demonstrated that the complex forms without the need for exogenous translocon machinery or chaperones, confirmed by negative stain electron microscopy. This work demonstrates the potential for using CFPS to express multi-subunit membrane-bound metalloenzymes directly into lipid bilayers.
Insights
Researchers developed a cell-free protein synthesis system to express particulate methane monooxygenase (pMMO), a key enzyme for converting methane to methanol, enabling future studies of its mechanism.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- Particulate methane monooxygenase (pMMO) is crucial for methanotrophic bacteria to oxidize methane to methanol.
- Studying pMMO is challenging due to the absence of a suitable recombinant expression system.
- This limitation hinders detailed mechanistic investigations and residue-specific studies via mutagenesis.
Purpose of the Study:
- To establish a novel cell-free protein synthesis (CFPS) system for in vitro expression of pMMO.
- To overcome the limitations of current expression systems for studying pMMO.
- To enable future investigations into the pMMO mechanism and enzyme engineering.
Main Methods:
- Developed an Escherichia coli lysate-based cell-free protein synthesis system.
- Utilized nanodiscs for in vitro expression of the multi-subunit pMMO complex.
- Employed a SUMO fusion construct for expressing the PmoB subunit and subsequent cleavage by Ulp1 protease.
- Used an affinity tag for isolating the complete pMMO complex.
- Confirmed complex formation using negative stain electron microscopy.
Main Results:
- Successfully expressed pMMO in vitro using the developed CFPS system.
- Demonstrated efficient cleavage of the SUMO fusion protein by Ulp1 protease.
- Confirmed the self-assembly of the complete pMMO complex within nanodiscs without exogenous factors.
- Verified the formation of the functional pMMO complex via electron microscopy.
Conclusions:
- The developed CFPS system provides a viable method for expressing multi-subunit membrane-bound metalloenzymes like pMMO.
- This in vitro expression system facilitates the study of pMMO structure and function.
- The findings pave the way for future research into methane oxidation mechanisms and enzyme engineering.
