Recombinant Enterobacteria phage RB70 Single-stranded DNA-binding protein (32)
Description
Biochemical Properties
Mechanistic insights:
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Truncation of the C-terminal domain (e.g., ΔPR201 variant) increases ssDNA affinity but reduces salt sensitivity .
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Zinc removal disrupts ssDNA binding, highlighting its role in maintaining structural integrity .
Recombinant Production and Applications
Recombinant RB70 gp32 is produced in E. coli or yeast systems (see for expression options). Commercial suppliers like Cusabio offer variants with tags (e.g., AviTag-Biotin) for pull-down assays or structural studies .
Notable findings:
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DNA chaperoning: Weak electron density in crystallography studies suggests ssDNA slides freely within the binding cleft, enabling sequence-independent interactions .
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Synergy with replicative enzymes: Enhances processivity of DNA polymerases and helicases by stabilizing ssDNA templates .
Research Implications
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Structural predictions: RB70 gp32 likely adopts an OB-fold (oligonucleotide/oligosaccharide-binding fold) similar to T4 gp32 and T7 gene 2.5 protein, as observed in phage Enc34 SSBs .
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Biotechnological utility: Used in in vitro replication systems and single-molecule studies to probe DNA-protein dynamics .
Unresolved Questions
Product Specs
Target Background
Q&A
Data Analysis and Contradiction Resolution
Q: How can researchers resolve contradictions in data when studying the effects of single-stranded DNA-binding proteins on DNA stability and PCR efficiency?
A: To resolve data contradictions:
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Replication: Repeat experiments multiple times to ensure consistency.
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Control Conditions: Include appropriate controls to isolate the effect of the protein.
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Statistical Analysis: Use statistical methods to compare results and assess significance.
Advanced Research Questions: Protein Engineering
Q: What strategies can be employed to engineer single-stranded DNA-binding proteins from bacteriophages for enhanced functionality or specificity?
A: Engineering strategies include:
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Site-Directed Mutagenesis: Alter specific amino acids to improve DNA binding affinity or specificity.
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Fusion Proteins: Create chimeric proteins by combining the DNA-binding domain with other functional domains (e.g., fluorescent tags) to enhance detection or recruitment capabilities .
Methodological Considerations for Phage-Derived Proteins
Q: How do researchers typically express and purify recombinant phage-derived proteins like single-stranded DNA-binding proteins?
A: Expression and purification typically involve:
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Host Selection: Choose an appropriate host organism (e.g., E. coli) for recombinant protein expression.
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Expression Vectors: Use vectors with suitable promoters and tags (e.g., His-tag) for efficient expression and purification.
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Purification Techniques: Employ methods like affinity chromatography or gel filtration to isolate the protein.
Applications in Molecular Biology
Q: What are some potential applications of single-stranded DNA-binding proteins from bacteriophages in molecular biology research?
A: Applications include:
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PCR Optimization: Enhance PCR efficiency by stabilizing single-stranded DNA.
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DNA Sequencing: Improve sequencing outcomes by minimizing secondary structures in DNA templates.
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Gene Delivery: Utilize phage-derived proteins in gene therapy vectors for targeted delivery.
Phage Receptor Binding Proteins and Their Role
Q: How do receptor binding proteins (RBPs) from bacteriophages interact with host cells, and what implications does this have for research?
A: RBPs mediate the initial attachment of phages to host bacteria by recognizing specific receptors on the bacterial surface . This interaction is crucial for phage infection and can be engineered for targeted drug delivery or diagnostic applications.
Bioinformatics and Genomic Analysis
Q: How can researchers use bioinformatics tools to analyze and annotate phage genomes, including those encoding single-stranded DNA-binding proteins?
A: Bioinformatics tools such as Phamerator and GenBank can be used to annotate phage genomes, predict gene functions, and compare genomic arrangements . This helps in understanding the genetic basis of phage-host interactions and identifying potential genes for recombinant protein production.
Targeted Delivery Systems
Q: How can bacteriophages and their proteins be engineered for targeted delivery systems in biomedical applications?
A: Phages can be engineered by modifying their surface proteins or genetic material to include targeting ligands or therapeutic moieties. This allows for targeted delivery of drugs or genes to specific cells, overcoming limitations in mammalian cell targeting .
Example Data Table: Experimental Conditions for Studying Single-Stranded DNA-Binding Proteins
| Experiment | Conditions | Expected Outcome |
|---|---|---|
| EMSA | 10 nM protein, 20 nM ssDNA, 20°C | Assess binding affinity |
| PCR | 100 ng protein/μL, 50 μL reaction | Enhance PCR efficiency |
| Structural Analysis | X-ray crystallography or NMR | Determine protein structure |
