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Isolation of mutants in M13 coat protein that affect its synthesis, processing, and assembly into phage
Abstract:
The major coat protein (gene 8 protein) of bacteriophage M13 has been studied intensively as a model of membrane assembly, protein packing, and protein-DNA interactions. Because this protein is essential for assembly of the phage, very few mutants have been isolated. We have therefore cloned the gene 8 into a plasmid under control of the araB promoter. In the presence of arabinose, the cloned gene is expressed at a rate comparable to that in an M13-infected cell. Plasmid-derived procoat is inserted across the plasma membrane and processed to coat at a normal rate. The coat can support plaque formation by a defective M13 virus (M13am8) with an amber mutation in its procoat gene. This complementation assay was used to screen the mutagenized, cloned gene 8 for mutants which fail to make fully functional coat. Mutants were obtained which fail to synthesize procoat, which do not convert procoat to mature coat protein, or in which the coat protein is incapable of assembling into infectious virions.
Insights
Researchers developed a new method to study bacteriophage M13 major coat protein mutants. This approach allows for the isolation and characterization of essential phage assembly proteins, advancing our understanding of viral structure and function.
Area of Science:
- Molecular Biology
- Virology
- Structural Biology
Background:
- Bacteriophage M13 major coat protein (gene 8) is crucial for phage assembly.
- Studying M13 coat protein mutants is challenging due to essentiality for phage viability.
- Previous research focused on M13 coat protein's role in membrane assembly and protein interactions.
Purpose of the Study:
- To develop a system for isolating and characterizing mutants of the M13 bacteriophage major coat protein.
- To investigate the functional domains of the M13 coat protein essential for phage assembly.
- To enable the study of protein-DNA interactions and membrane assembly using M13 coat protein.
Main Methods:
- Cloning the M13 gene 8 into a plasmid under the control of the araB promoter for inducible expression.
- Utilizing a complementation assay with a defective M13 virus (M13am8) to screen for functional coat protein mutants.
- Introducing mutations into the cloned gene 8 and analyzing the resulting procoat processing and assembly.
Main Results:
- Successfully expressed functional M13 major coat protein from a plasmid system at levels comparable to phage infection.
- Identified mutants with defects in procoat synthesis, processing to mature coat protein, or assembly into infectious virions.
- Demonstrated that plasmid-derived coat protein can support plaque formation by M13am8, validating the complementation assay.
Conclusions:
- The developed plasmid-based expression system and complementation assay are effective for isolating and studying M13 major coat protein mutants.
- This system facilitates detailed investigation into the structure-function relationships of the M13 coat protein.
- The findings provide new insights into bacteriophage assembly, membrane protein insertion, and protein-DNA interactions.