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Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy
Published on: September 17, 2017
Cell-free protein synthesis technology in NMR high-throughput structure determination
Shin-ichi Makino1, Michael A Goren, Brian G Fox
1Department of Biochemistry, Center for Eukaryotic Structural Genomics, College of Agricultural and Life Sciences, University of Wisconsin, Madison, WI, USA.
Methods in Molecular Biology (Clifton, N.J.)
|March 6, 2010
Summary
This study details a cell-free protein production platform for generating stable isotope-labeled eukaryotic proteins. The methods enable efficient screening and production for NMR structure determination, including for membrane proteins.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Biology
Background:
- Nuclear Magnetic Resonance (NMR) structure determination requires substantial quantities of pure, stable isotope-labeled proteins.
- Eukaryotic protein expression can be challenging, necessitating efficient and scalable production platforms.
- The Center for Eukaryotic Structural Genomics (CESG) has developed a cell-free translation system to address these challenges.
Purpose of the Study:
- To describe the implementation of the CESG cell-free translation platform for producing eukaryotic proteins.
- To provide practical protocols for small-scale screening and large-scale production of stable isotope-labeled proteins for NMR studies.
- To present methods for characterizing protein expression, solubility, and purification, and for producing integral membrane proteins.
Main Methods:
- Utilizing wheat germ cell-free translation for small-scale screening of protein expression, solubility, and purification success.
- Employing robotic automation for small-scale cell-free translation, large-scale protein production, and automated purification of His(6)-tagged proteins.
- Developing protocols for producing integral membrane proteins in the presence of detergents or unilamellar liposomes.
Main Results:
- Small-scale cell-free reactions serve as effective predictors for large-scale protein production success.
- Sufficient protein quantities for bioanalytical and functional characterizations are often obtained from small-scale reactions.
- Protocols facilitate the integration of isotopically labeled proteins into NMR structure determination workflows.
Conclusions:
- The described cell-free translation platform provides an efficient method for producing eukaryotic proteins for structural biology.
- The integrated robotic systems enhance throughput and automation in protein production and purification.
- The developed protocols are applicable to a wide range of proteins, including challenging integral membrane proteins, for NMR structure determination.
