Recombinant Chromobacterium violaceum UPF0250 protein CV_3095 (CV_3095)

What is CV_3095 and what organism does it come from?

CV_3095 is a small protein (89 amino acids, 10.156 kDa) belonging to the UPF0250 family that is encoded by the Chromobacterium violaceum genome . The specific strain reference is Chromobacterium violaceum (strain ATCC 12472 / DSM 30191 / JCM 1249 / NBRC 12614 / NCIMB 9131 / NCTC 9757) . Chromobacterium violaceum is a gram-negative bacterium abundantly found in soil and water microbiota of tropical and subtropical regions worldwide . This organism has gained significant research interest as it has emerged as an important model of an environmental opportunistic pathogen with high virulence in human infections and mouse infection models .

What is currently known about the function of UPF0250 family proteins?

The UPF0250 family, to which CV_3095 belongs, is categorized as a family of proteins with unknown function (UPF) . This designation indicates that while these proteins have been identified and sequenced across multiple organisms, their specific biological roles remain largely uncharacterized.

Research approaches to determine the function of such uncharacterized proteins typically include:

• Comparative genomic analyses with better-characterized organisms

• Gene knockout studies to observe phenotypic changes

• Protein-protein interaction studies to identify binding partners

• Structural analyses to identify potential functional domains

• Expression studies under various conditions to determine when the protein is produced

What are the optimal conditions for recombinant expression of CV_3095?

Based on established protocols for similar proteins, the following methodology would be suitable for CV_3095 expression:

Expression System Selection:
E. coli is the recommended expression system for CV_3095 due to its cost-effectiveness and efficiency for small, soluble bacterial proteins . Consider using BL21(DE3) or Rosetta strains, with the latter being particularly useful if CV_3095 contains rare codons that might cause ribosomal stalling .

Vector Selection:
A pET-based expression vector with a fusion tag would be optimal. Consider using a maltose-binding protein (MBP) tag, which has been shown to dramatically boost expression of challenging proteins and improve solubility . The vector should include:

• A strong, inducible promoter (T7)

• An appropriate fusion tag (MBP or 6xHis)

• A precision protease cleavage site for tag removal

Culture Conditions:

• Temperature: 18-25°C for induction phase to minimize inclusion body formation

• Medium: Enriched media such as Terrific Broth (TB) or 2xYT

• Induction: 0.1-0.5 mM IPTG when culture reaches OD600 of 0.6-0.8

• Post-induction growth: 16-18 hours at reduced temperature (18°C)

Optimization Parameters:
If initial expression yields are low, consider optimizing:

• Codon usage (through codon-optimized synthetic genes)

• Introduction of mutations to disrupt potential ribosomal stalling proline-rich motifs

• Adjusting induction parameters (IPTG concentration, temperature, duration)

• Testing different fusion tags (SUMO, GST, TrxA) if MBP is not effective

What purification strategy is most effective for obtaining high-purity CV_3095?

For efficient purification of recombinant CV_3095, the following multi-step strategy is recommended:

Step 1: Cell Lysis and Initial Clarification

• Resuspend cell pellet in lysis buffer containing:

  • 50 mM Tris-HCl pH 8.0

  • 300 mM NaCl

  • 1 mM DTT

  • Protease inhibitor cocktail

  • 0.1% Triton X-100 (to reduce aggregation)

• Lyse cells via sonication or high-pressure homogenization

• Clarify lysate by centrifugation (20,000 × g, 30 min, 4°C)

Step 2: Ammonium Sulfate Precipitation

• Add ammonium sulfate to 40-60% saturation to precipitate the majority of protein contaminants while keeping CV_3095 in solution

• Collect supernatant after centrifugation (15,000 × g, 20 min, 4°C)

Step 3: Affinity Chromatography

• If using MBP-tagged CV_3095:

  • Apply supernatant to MBP-Trap column

  • Wash extensively with binding buffer

  • Elute with 10 mM maltose

• If using His-tagged CV_3095:

  • Apply supernatant to Ni-NTA column

  • Wash with increasing imidazole concentrations (10-40 mM)

  • Elute with 250 mM imidazole

Step 4: Tag Removal (Optional)

• Incubate with appropriate protease (TEV, PreScission, etc.)

• Remove cleaved tag by reverse affinity chromatography

Step 5: Heparin Affinity Chromatography

• Apply protein to heparin column to remove remaining protein contaminants, truncated proteins, and nucleic acid contamination

• Elute with a gradient of increasing NaCl concentration

Step 6: Size Exclusion Chromatography (Polishing Step)

• Apply concentrated protein to Superdex 75 or similar column

• Collect fractions containing pure CV_3095

• Analyze purity by SDS-PAGE and Western blot

Expected yield: 1-9 mg of pure protein from 2 L of bacterial culture , with homogeneity >90% as assessed by SDS-PAGE.

How can I verify the identity and functionality of purified recombinant CV_3095?

Verification of purified recombinant CV_3095 should include multiple analytical approaches:

Identity Verification:

• Mass Spectrometry Analysis:

  • Perform intact mass analysis to confirm the theoretical mass (10.156 kDa)

  • Conduct peptide mass fingerprinting after tryptic digest

  • Compare observed peptides with in silico digest of theoretical sequence

• N-terminal Sequencing:

  • Confirm the first 5-10 amino acids using Edman degradation

  • Verify proper processing of any signal peptide if present

• Western Blot Analysis:

  • Use anti-His or anti-MBP antibodies (depending on tag used)

  • Consider developing antibodies against CV_3095 for future studies

Structural Integrity Assessment:

• Circular Dichroism (CD) Spectroscopy:

  • Analyze secondary structure elements

  • Compare with CD profiles of related proteins if available

• Thermal Shift Assay:

  • Determine protein stability and melting temperature

  • Useful for buffer optimization and storage condition determination

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS):

  • Determine oligomeric state and homogeneity

  • Verify absence of aggregation

Functional Characterization:
Since CV_3095 is an uncharacterized protein, functional assays may need to be developed based on:

• Protein-Protein Interaction Studies:

  • Pull-down assays with C. violaceum lysate

  • Yeast two-hybrid screening

  • Surface plasmon resonance with potential binding partners

• Enzymatic Activity Screening:

  • Test for common enzymatic activities (phosphatase, kinase, protease, etc.)

  • Substrate profiling if enzymatic function is suspected

• Structural Studies:

  • X-ray crystallography or NMR studies for detailed structural information

  • In silico structural prediction and comparison with functionally characterized proteins

What are the key considerations for designing experiments with CV_3095?

When designing experiments involving recombinant CV_3095, researchers should consider several critical factors to ensure meaningful and reproducible results:

Experimental Design Framework:

• Define Clear Research Questions:

  • Formulate specific hypotheses about CV_3095 function

  • Design experiments with appropriate positive and negative controls

  • Consider pilot studies to optimize experimental conditions

• Protein Stability Considerations:

  • Determine optimal buffer conditions (pH, salt concentration, additives)

  • Assess thermal stability in different buffers using thermal shift assays

  • Evaluate freeze-thaw stability and long-term storage conditions

  • Consider addition of reducing agents if cysteine residues are present

• Concentration and Stoichiometry:

  • Determine working concentration ranges based on estimated physiological levels

  • Consider protein concentration effects on oligomerization state

  • Calculate proper molar ratios for interaction studies

Methodological Approaches:

• Functional Annotation Strategies:

  • Genomic context analysis (examining neighboring genes)

  • Phylogenetic profiling (correlation of gene presence across species)

  • Structural homology modeling to predict function

• Localization Studies:

  • Generate fluorescent protein fusions for cellular localization in heterologous systems

  • Perform subcellular fractionation studies

  • Create antibodies against CV_3095 for immunolocalization

• Interaction Network Mapping:

  • Conduct pull-down assays coupled with mass spectrometry

  • Perform bacterial two-hybrid screens

  • Consider proximity labeling approaches (BioID, APEX)

• Phenotypic Analysis:

  • Generate knockout or knockdown strains of CV_3095 in C. violaceum

  • Assess phenotypic changes in growth, stress response, and virulence

  • Perform complementation studies to confirm phenotype specificity

• Structural Biology Approaches:

  • Optimize conditions for crystal formation or NMR studies

  • Consider protein engineering to improve crystallization properties

  • Perform in silico structure prediction and validation

How should I design controls for experiments involving CV_3095?

Proper experimental controls are essential for robust data interpretation in studies involving CV_3095:

Negative Controls:

• Empty Vector Control:

  • Cells expressing the same vector backbone without the CV_3095 insert

  • Processed identically to experimental samples

  • Controls for effects of the expression system itself

• Inactive Protein Variant:

  • If functional domains or critical residues are identified, create point mutants

  • Use heat-denatured CV_3095 as a control for activity assays

  • Consider scrambled peptide controls for binding studies

• Unrelated Protein Control:

  • Use an unrelated protein of similar size and properties

  • Ideally another UPF family protein that lacks the hypothesized function

  • Processed using identical purification methods

Positive Controls:

• Known Functional Homologs:

  • Include characterized proteins from the UPF0250 family if available

  • Use proteins with similar predicted structures or domains

  • Consider proteins with established functions in C. violaceum

• Validated Interaction Partners:

  • If binding partners are identified, include known positive interactors

  • Use synthetic peptides corresponding to binding regions

  • Include established substrate analogs for enzymatic assays

Technical Controls:

• Reproducibility Controls:

  • Perform biological replicates (different protein preparations)

  • Conduct technical replicates (multiple analyses of the same preparation)

  • Include internal standards for quantitative assays

• System Validation Controls:

  • Use well-characterized protein pairs to validate interaction assays

  • Include enzyme/substrate pairs with known kinetics for activity assays

  • Develop standard curves for quantitative measurements

• Contaminant Controls:

  • Test for the effects of potential contaminants (e.g., endotoxin)

  • Verify tag-only controls do not exhibit the observed activities

  • Perform mock purifications from non-expressing cells

How might CV_3095 be involved in C. violaceum pathogenicity?

Although the exact role of CV_3095 in Chromobacterium violaceum pathogenicity is not explicitly described in the search results, we can propose several hypotheses based on the known pathogenicity mechanisms of C. violaceum:

Potential Connections to Virulence Mechanisms:

• Type III Secretion System (T3SS) Interactions:
  C. violaceum possesses two T3SSs, with the Cpi-1 T3SS playing a pivotal role in host cell interactions . CV_3095 could potentially:

  • Function as a chaperone for T3SS effector proteins

  • Act as a regulatory protein controlling T3SS gene expression

  • Serve as an accessory protein in the T3SS machinery assembly

  • Participate in host cell recognition or adhesion processes

• Inflammasome Interaction:
  The Cpi-1 T3SS is recognized by the NLRC4 inflammasome in innate immune cells . CV_3095 might:

  • Modulate inflammatory responses by interfering with inflammasome assembly

  • Contribute to evasion of immune recognition

  • Participate in processes related to pyroptosis, which is a key event in C. violaceum clearance

• Stress Response and Adaptation:
  Small bacterial proteins often function in stress response pathways. CV_3095 could:

  • Facilitate adaptation to host environments

  • Participate in response to oxidative stress during infection

  • Enable survival under nutrient limitation within host cells

Experimental Approaches to Test These Hypotheses:

• Gene Knockout Studies:

  • Generate CV_3095 deletion mutants in C. violaceum

  • Assess virulence in mouse infection models

  • Evaluate survival within macrophages or epithelial cells

  • Measure T3SS function in the absence of CV_3095

• Protein Localization:

  • Determine if CV_3095 co-localizes with T3SS components

  • Assess whether CV_3095 is secreted during infection

  • Examine distribution during different stages of infection

• Interaction Studies:

  • Perform pull-down assays with T3SS components

  • Conduct yeast two-hybrid screens with host proteins

  • Use bacterial two-hybrid systems to map bacterial protein interactors

• Transcriptional Analysis:

  • Compare gene expression profiles between wild-type and CV_3095 mutants

  • Identify co-regulated genes during infection

  • Determine if CV_3095 expression changes during infection progression

What structural analysis techniques should be considered for studying CV_3095?

Given the small size (89 amino acids, 10.156 kDa) of CV_3095, several structural biology techniques are particularly suitable:

X-ray Crystallography:

• Advantages: High-resolution structural information, ability to see bound ligands

• Approach:

  • Screen multiple crystallization conditions (sparse matrix approach)

  • Optimize promising conditions for crystal quality

  • Consider crystallizing with potential binding partners

  • Use MBP or other fusion tags as crystallization chaperones if direct crystallization fails

  • Employ selenomethionine incorporation for phase determination

Nuclear Magnetic Resonance (NMR) Spectroscopy:

• Advantages: Solution structure, dynamic information, direct binding studies

• Particularly suitable for CV_3095 due to its small size (optimal for proteins <20 kDa)

• Approach:

  • Express 15N and 13C labeled protein for backbone and side-chain assignments

  • Collect HSQC, NOESY, and TOCSY experiments

  • Determine solution structure

  • Study protein dynamics through relaxation measurements

  • Perform titration experiments with potential binding partners

Cryo-Electron Microscopy (Cryo-EM):

• Generally less suitable for proteins <50 kDa unless part of a larger complex

• Consider if CV_3095 forms large oligomers or complexes with other proteins

Complementary Biophysical Techniques:

• Small-Angle X-ray Scattering (SAXS):

  • Provides low-resolution envelope of protein in solution

  • Useful for studying oligomerization and conformational changes

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Maps solvent-accessible regions and binding interfaces

  • Provides information on protein dynamics and conformational changes

• Circular Dichroism (CD):

  • Assesses secondary structure composition

  • Monitors structural changes under different conditions

• Molecular Dynamics (MD) Simulations:

  • Predicts dynamic behavior and conformational changes

  • Identifies potential binding pockets and functional regions

Structural Prediction and Analysis:

• Employ AlphaFold2 or RoseTTAFold for initial structural predictions

• Use predicted structures to guide experimental design

• Perform conservation mapping across UPF0250 family members to identify functionally important residues

How can I investigate potential protein-protein interactions of CV_3095?

Investigating protein-protein interactions (PPIs) of CV_3095 requires a comprehensive approach combining multiple techniques:

In Vitro Interaction Methods:

• Pull-down Assays:

  • Immobilize tagged CV_3095 as bait protein

  • Incubate with C. violaceum lysate or purified potential partners

  • Identify binding partners by mass spectrometry

  • Validate interactions with reciprocal pull-downs

• Surface Plasmon Resonance (SPR):

  • Immobilize CV_3095 on sensor chip

  • Flow potential binding partners over the surface

  • Determine binding kinetics (kon, koff) and affinity (KD)

  • Perform competition experiments to map binding sites

• Isothermal Titration Calorimetry (ITC):

  • Directly measure thermodynamic parameters of binding

  • Determine stoichiometry, binding constant, enthalpy, and entropy

  • No immobilization required, both proteins in solution

• Microscale Thermophoresis (MST):

  • Label CV_3095 with fluorescent dye

  • Titrate with potential binding partners

  • Measure changes in thermophoretic mobility

  • Suitable for weak and transient interactions

In Vivo Interaction Methods:

• Bacterial Two-Hybrid System:

  • Fuse CV_3095 to DNA-binding domain

  • Create library of prey proteins fused to activation domain

  • Screen for reporter gene activation indicating interaction

  • Particularly suitable for bacterial protein interactions

• Proximity-Based Labeling:

  • Fuse CV_3095 to BioID or APEX2 enzymes

  • Express in C. violaceum or heterologous system

  • Identify proximal proteins through biotinylation and pulldown

  • Useful for capturing weak or transient interactions

• Co-immunoprecipitation (Co-IP):

  • Express tagged CV_3095 in C. violaceum

  • Precipitate with tag-specific antibodies

  • Identify co-precipitating proteins by mass spectrometry

  • Validate with reciprocal Co-IPs

Computational Prediction Methods:

• Interactome Prediction:

  • Use tools like STRING, STITCH, or PrePPI

  • Predict interactions based on genomic context, co-expression, and co-evolution

  • Prioritize candidates for experimental validation

• Docking Studies:

  • Perform in silico docking with potential partners

  • Evaluate binding energy and interface complementarity

  • Guide mutagenesis studies to validate interactions

• Interface Prediction:

  • Identify potential protein-protein interaction sites

  • Target conserved surface patches for mutagenesis

  • Design peptides to disrupt predicted interactions

Interaction Validation Strategies:

• Mutagenesis:

  • Introduce point mutations at predicted interface residues

  • Assess effect on binding using methods above

  • Perform alanine scanning to map critical residues

• Domain Mapping:

  • Create truncation variants of CV_3095

  • Determine minimal binding regions

  • Design peptide competitors based on binding regions

How can I troubleshoot poor expression or solubility of recombinant CV_3095?

Troubleshooting poor expression or solubility of recombinant CV_3095 requires a systematic approach to identify and address specific issues:

Expression Troubleshooting:

Solubility Troubleshooting:

Advanced Strategies:

• Fusion Tag Optimization:

  • Test multiple fusion tags (MBP, SUMO, GST, TRX)

  • Vary tag position (N-terminal vs. C-terminal)

  • Include flexible linkers between tag and CV_3095

• Expression Strain Engineering:

  • Test multiple E. coli strains (BL21, C41/C43, SHuffle)

  • Consider expression in non-E. coli systems if persistent issues

  • Use strains with enhanced disulfide bond formation if necessary

• Lysis Buffer Optimization:

  • Test additives that enhance solubility:

    • Sugars (glucose, sucrose)

    • Amino acids (arginine, glutamate)

    • Osmolytes (betaine, TMAO)

    • Non-detergent sulfobetaines (NDSB-201)

• Refolding Strategies:

  • If inclusion bodies persist, develop refolding protocol:

    • Solubilize in 8M urea or 6M guanidine-HCl

    • Remove denaturant by dialysis or dilution

    • Add redox pairs for disulfide formation if needed

    • Use artificial chaperones to assist folding

What are common analytical issues when working with small proteins like CV_3095?

Working with small proteins like CV_3095 (89 amino acids, 10.156 kDa) presents unique analytical challenges:

SDS-PAGE Analysis Challenges:

Mass Spectrometry Challenges:

Size Exclusion Chromatography Challenges:

Western Blot Challenges:

Advanced Analytical Approaches:

• Native MS for Oligomeric State:

  • Maintain non-covalent interactions during ionization

  • Determine accurate masses of complexes

  • Assess binding of small molecules or metal ions

• Ion-Mobility MS for Conformation:

  • Separate protein conformers based on shape

  • Detect subtle structural changes

  • Complement with hydrogen-deuterium exchange

• Capillary Electrophoresis:

  • High-resolution separation based on charge/size

  • Minimal sample consumption

  • Can be coupled to MS for further analysis

• Analytical Ultracentrifugation:

  • Direct measurement of molecular weight

  • Determination of oligomeric states

  • Analysis of shape parameters

How should I address reproducibility issues in CV_3095 experiments?

Ensuring reproducibility in experiments involving CV_3095 requires careful attention to multiple factors:

Protein Production Reproducibility:

Experimental Design Reproducibility:

Data Analysis Reproducibility:

Systematic Approach to Reproducibility:

• Quality Control Measures:

  • Develop specific QC assays for CV_3095:

    • Activity assay (once function is determined)

    • Thermal stability (Tm) measurement

    • SEC profile for aggregation assessment

  • Establish acceptance criteria for experiments

  • Maintain reference standards from successful preparations

• Experimental Design Practices:

  • Use randomization to minimize bias

  • Include internal references across experiments

  • Blind analysis when possible

  • Perform power calculations to determine sample size

• Laboratory Information Management:

  • Use electronic lab notebooks with version control

  • Implement sample tracking systems

  • Store metadata with experimental results

  • Document all environmental conditions

• Cross-validation Approaches:

  • Verify key findings with orthogonal methods

  • Test reproducibility across different operators

  • Consider independent replication in collaborating labs