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Published on: September 16, 2011
An in vitro screening system for protein splicing inhibitors based on green fluorescent protein as an indicator
Jaya Pal Gangopadhyay1, Shu-Qin Jiang, Henry Paulus
1Boston Biomedical Research Institute, Watertown, Massachusetts 02472, USA.
Abstract:
This paper describes an in vitro fluorometric assay system for protein splicing based on the RecA intein of Mycobacterium tuberculosis and a modified green fluorescent protein (GFP). The assay takes advantage of the fact that polypeptides inserted adjacent to residue 129 of GFP cause the protein to form inclusion bodies when expressed in Escherichia coli and to be incapable of fluorophore formation. However, when the inserted polypeptide is an intein, the renatured fusion protein can undergo protein splicing and chromophore formation. Comparison of chromophore formation by renatured GFP-intein fusion and renatured GFP showed that under optimal conditions (pH 6.5 and 20 degrees C) protein splicing is significantly slower than GFP chromophore formation. Taking advantage of the reversible inhibition of protein splicing by zinc ion, a fluorometric protein splicing assay was developed in which the denatured fusion protein of GFP and the RecA intein was purified on a metal ion affinity column and renatured in the presence of 2 mM ZnCl2. When diluted into appropriate buffers, protein splicing could be initiated by the addition of a molar excess of EDTA and followed fluorometrically. This assay should be valuable as a high-throughput screening system for protein splicing inhibitors as potential antimycobacterial agents and as tools for studying the mechanism of protein splicing.
Insights
A novel fluorometric assay system was developed for protein splicing using a Mycobacterium tuberculosis RecA intein and green fluorescent protein (GFP). This assay enables high-throughput screening for potential antimycobacterial agents by monitoring protein splicing activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- Protein splicing is a post-translational modification.
- Inteins mediate protein splicing.
- Developing efficient assays for protein splicing is crucial for drug discovery.
Purpose of the Study:
- To develop an in vitro fluorometric assay system for protein splicing.
- To utilize the RecA intein from Mycobacterium tuberculosis and a modified green fluorescent protein (GFP).
- To establish a high-throughput screening system for potential antimycobacterial agents.
Main Methods:
- A fusion protein of GFP and the RecA intein was constructed.
- The fusion protein was expressed in E. coli, causing inclusion body formation.
- Protein splicing was induced upon renaturation and monitored fluorometrically using zinc ion inhibition and EDTA activation.
Main Results:
- The developed assay system successfully monitored protein splicing in vitro.
- Protein splicing kinetics were characterized under optimal conditions (pH 6.5, 20°C).
- The assay demonstrated the feasibility of using reversible zinc ion inhibition for controlling and initiating splicing.
Conclusions:
- The novel fluorometric assay is effective for studying protein splicing.
- This system serves as a valuable tool for high-throughput screening of protein splicing inhibitors.
- The assay has potential applications in identifying novel antimycobacterial agents and understanding protein splicing mechanisms.
