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Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli
Published on: January 6, 2015
Heterologous gene expression in a membrane-protein-specific system
G J Turner1, R Reusch, A M Winter-Vann
1Department of Physiology & Biophysics, University of Miami School of Medicine, Miami, Florida, 33101, USA. gturner@chroma.med.miami.edu
Protein Expression and Purification
|November 5, 1999
Summary
Researchers developed a novel expression system using Halobacterium salinarum to produce heterologous proteins. This system achieved high-level expression of membrane proteins, demonstrating its potential for significant protein production.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- High-level production of heterologous proteins is crucial for research and therapeutic applications.
- Existing expression systems face limitations in producing complex membrane proteins.
- Halobacterium salinarum's bacteriorhodopsin production machinery offers a potential alternative.
Purpose of the Study:
- To construct and characterize a novel heterologous protein expression system in Halobacterium salinarum.
- To identify molecular determinants within the bacterio-opsin gene critical for high-level expression.
- To assess the system's capacity for producing milligram quantities of membrane-associated proteins.
Main Methods:
- Assembled cloning vectors fusing the bacterio-opsin gene (bop) to heterologous genes.
- Transferred gene fusions into H. salinarum expression vectors.
- Characterized expression of various gene fusions, including receptor proteins and enzyme subunits.
Main Results:
- Identified previously undescribed molecular determinants in the bop gene essential for high-level expression.
- Demonstrated that C-terminal tags significantly decrease bop gene mRNA and protein accumulation.
- Achieved expression of a bacteriorhodopsin-aspartate transcarbamylase fusion protein at 7 mg/L.
Conclusions:
- A novel heterologous protein expression system in H. salinarum has been developed.
- The bop gene contains critical elements for high-level protein production.
- This system shows promise for producing milligram quantities of membrane proteins.
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