Recombinant Gloeobacter violaceus 30S ribosomal protein S19 (rpsS)
Description
Introduction
Gloeobacter violaceus is a primitive cyanobacterium known for its unique photosynthetic and respiratory systems . Ribosomal proteins, including the 30S ribosomal protein S19 (rpsS), are essential components of the ribosome, which is responsible for protein synthesis in all living cells . Recombinant production of Gloeobacter violaceus 30S ribosomal protein S19 (rpsS) involves synthesizing the protein in a host organism, often Escherichia coli, for research and industrial applications .
Background on Gloeobacter violaceus
Gloeobacter violaceus is a distinctive cyanobacterium because it lacks thylakoid membranes, which are typically found in other cyanobacteria and are the site of photosynthesis . Instead, photosynthesis occurs in the plasma membrane . This structural simplicity makes Gloeobacter violaceus an interesting subject for studying the evolution of photosynthesis and the functional organization of its cellular components .
Role of 30S Ribosomal Protein S19 (rpsS)
The 30S ribosomal subunit is a component of the bacterial ribosome that binds to messenger RNA (mRNA) and facilitates the translation of genetic code into proteins. The S19 protein is a structural component of the 30S subunit, crucial for its assembly and function .
Recombinant Production of rpsS
Recombinant protein production involves cloning the gene encoding the protein of interest into an expression vector, which is then introduced into a host organism like E. coli . The host organism then produces the protein, which can be purified for downstream applications .
4.1. Process
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Gene Cloning: The rpsS gene from Gloeobacter violaceus is isolated and inserted into a plasmid vector .
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Transformation: The plasmid is introduced into E. coli cells .
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Expression: E. coli cells are cultured under conditions that induce rpsS gene expression .
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Purification: The recombinant RpsS protein is isolated from the E. coli lysate using affinity chromatography or other purification techniques .
4.2. Benefits
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High Yield: Recombinant production allows for high yields of the target protein compared to direct extraction from the native organism .
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Purity: Recombinant proteins can be purified to high homogeneity, which is essential for structural and functional studies .
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Modifications: Recombinant techniques allow for the introduction of specific mutations or tags to facilitate protein detection and purification .
Research Applications
Recombinant Gloeobacter violaceus 30S ribosomal protein S19 (rpsS) is useful in various research applications:
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Structural Studies: Recombinant RpsS can be used for X-ray crystallography or NMR spectroscopy to determine its three-dimensional structure and understand its interactions within the 30S ribosomal subunit.
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Functional Assays: The purified protein can be used in in vitro assays to study its role in ribosome assembly, mRNA binding, and translation fidelity.
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Drug Discovery: RpsS is a potential target for developing new antibiotics. Understanding its structure and function can aid in designing drugs that specifically inhibit bacterial protein synthesis.
Genomic Insights
Genomic analysis of Gloeobacter violaceus and related species reveals insights into the evolution and function of ribosomal proteins. For example, studies on endophytic Streptomyces species have identified biosynthetic gene clusters (BGCs) responsible for producing antimicrobial compounds, including non-ribosomal peptides and ribosomally synthesized peptides . While not directly related to RpsS, these studies highlight the importance of understanding the genetic context and interactions of ribosomal proteins in bacteria .
Data Tables
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Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your preferred format during order placement for customized preparation.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires prior arrangement and incurs additional charges.
The tag type is determined during production. If you require a specific tag, please inform us, and we will prioritize its development.
Target Background
Protein S19 forms a complex with S13, exhibiting strong binding affinity to the 16S ribosomal RNA.
KEGG: gvi:gvip122
STRING: 251221.gvip122
Q&A
Basic Research Questions
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What is the optimal expression system for producing recombinant Gloeobacter violaceus 30S ribosomal protein S19?
Based on established protocols for other cyanobacterial ribosomal proteins, E. coli represents the most effective heterologous expression system for recombinant Gloeobacter violaceus ribosomal proteins . When expressing S19, researchers should consider cloning the rpsS gene into an appropriate expression vector, followed by transformation into E. coli host strains optimized for protein expression. The expression conditions should include IPTG induction at mid-log phase, with growth temperatures between 18-30°C to balance protein yield and solubility. Purification typically involves a combination of affinity chromatography (using histidine or other fusion tags), followed by size-exclusion chromatography to ensure high purity (>85% as determined by SDS-PAGE) .
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What storage conditions ensure maximum stability and activity of recombinant S19 protein?
The stability of recombinant ribosomal proteins is significantly influenced by storage conditions. Based on documented protocols for similar ribosomal proteins like S10, recombinant S19 should be stored according to the following guidelines:
| Storage Form | Temperature | Shelf Life | Additional Recommendations |
|---|---|---|---|
| Liquid | -20°C/-80°C | 6 months | Add glycerol (5-50% final concentration) |
| Lyophilized | -20°C/-80°C | 12 months | Reconstitute in deionized sterile water |
| Working aliquots | 4°C | Up to 1 week | Avoid repeated freezing and thawing |
For reconstitution of lyophilized protein, it is recommended to briefly centrifuge the vial before opening to bring contents to the bottom, then reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL . Addition of glycerol (recommended final concentration 50%) followed by aliquoting is advised for long-term storage to maintain protein stability and prevent degradation .
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How can researchers verify the structural integrity and purity of recombinant S19 protein preparations?
Multiple analytical methods should be employed to verify structural integrity and purity of recombinant S19 preparations:
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SDS-PAGE analysis: Provides initial assessment of protein purity, with expected purity levels >85%
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Mass spectrometry: Confirms protein identity and integrity through accurate mass determination
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Circular dichroism spectroscopy: Evaluates secondary structure elements and proper folding
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Size-exclusion chromatography: Assesses aggregation state and homogeneity
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Limited proteolysis: Probes for correctly folded domains through resistance to enzymatic digestion
When analyzing recombinant S19, researchers should also consider functional assays that test the protein's ability to participate in ribosomal assembly, as the ultimate verification of native-like properties.
