Recombinant UPF0232 protein Mb0004 (Mb0004)

What is UPF0232 protein Mb0004 and what organism does it originate from?

UPF0232 protein Mb0004 is a hypothetical protein belonging to the UPF0150 protein family from Mycobacterium bovis (strain ATCC BAA-935 / AF2122/97). The protein is encoded by the gene BQ2027_MB0004 and has been cataloged in the UniProt database with the accession number Q7U313 . UPF (Uncharacterized Protein Family) designation indicates that while the protein has been identified through genomic sequencing, its biological function remains largely unknown. Similar proteins have been identified in other mycobacterial species, including Mycobacterium ulcerans (UPF0232 protein MUL_0004) .

What expression systems are available for recombinant production of UPF0232 protein Mb0004?

The recombinant UPF0232 protein Mb0004 has been successfully expressed in two main systems:

• E. coli expression system: This bacterial expression system (product code CSB-EP742566MVH-B) is widely used for basic research applications and provides good yields for structural and functional studies .

• Yeast expression system: For applications requiring eukaryotic post-translational modifications, a yeast-based expression system (product code CSB-YP742566MVH) is available .

Both expression systems produce the full-length protein (amino acids 1-187) with purity levels typically greater than 85% as determined by SDS-PAGE analysis . The choice between these systems should be guided by the specific requirements of your research, particularly if post-translational modifications are relevant to your study.

What purification strategies are most effective for recombinant UPF0232 protein Mb0004?

Based on established protocols for similar recombinant proteins, an effective purification strategy for UPF0232 protein Mb0004 would include:

• Initial Clarification: Cell disruption by sonication in 20 mM Tris-HCl buffer (pH 8.0) containing 50 mM NaCl, followed by centrifugation to remove cell debris .

• Heat Treatment: Taking advantage of the thermostability of mycobacterial proteins, heat treatment at 70°C for 30 minutes can effectively remove many E. coli host proteins .

• Column Chromatography: Sequential purification using:

  • Immobilized metal affinity chromatography (IMAC) using the His-tag

  • Ion exchange chromatography (Resource Q column)

  • Size exclusion chromatography (Superdex 75)

• Buffer Exchange: Final exchange into storage buffer (20 mM Tris-HCl pH 8.0) .

This protocol typically yields protein with >90% purity suitable for structural and functional studies. The exact conditions may need optimization for maximum yield and activity specific to UPF0232 protein Mb0004.

What are the recommended storage conditions for maintaining stability of purified recombinant UPF0232 protein Mb0004?

For optimal stability of recombinant UPF0232 protein Mb0004, the following storage recommendations apply:

• Short-term storage (up to one week): Store working aliquots at 4°C in buffer containing 20 mM Tris-HCl pH 8.0 .

• Long-term storage:

  • Lyophilized form: Stable for up to 12 months at -20°C/-80°C .

  • Liquid form: Add glycerol to a final concentration of 5-50% (typically 50%) and store at -20°C/-80°C for up to 6 months .

• Reconstitution of lyophilized protein: Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Avoiding degradation: Repeated freeze-thaw cycles should be avoided. It is recommended to prepare small working aliquots for single use .

The shelf life is influenced by multiple factors including storage state, buffer components, storage temperature, and the intrinsic stability of the protein itself.

What is known about the three-dimensional structure of UPF0232 protein Mb0004?

While the precise three-dimensional structure of UPF0232 protein Mb0004 has not been experimentally determined, structural insights can be inferred from related proteins:

• Homology modeling: Based on the structure of the homologous UPF0232 protein MUL_0004 from Mycobacterium ulcerans (which has a computed structure model in AlphaFold DB with a global pLDDT score of 74.96), UPF0232 protein Mb0004 likely adopts a similar fold .

• Predicted structural features: The protein likely contains a mixture of α-helical and β-sheet secondary structural elements, possibly in an α-β-β-β-α arrangement similar to that observed in TTHA0281, a hypothetical protein from Thermus thermophilus that belongs to the UPF0150 family .

• Quaternary structure: Some UPF family proteins form homotetramers, which might also be the case for UPF0232 protein Mb0004, though this requires experimental verification .

For definitive structural determination, researchers should consider X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, or cryo-electron microscopy techniques, which would provide atomic-level resolution of the protein structure.

What methodologies are recommended for investigating the structural properties of UPF0232 protein Mb0004?

To thoroughly investigate the structural properties of UPF0232 protein Mb0004, researchers should consider a multi-method approach:

• X-ray Crystallography:

  • Crystallization screening using various precipitation agents (PEG, ammonium sulfate)

  • Data collection at synchrotron radiation facilities

  • Structure determination by molecular replacement using homologous structures or by experimental phasing methods such as MAD (Multiple-wavelength Anomalous Dispersion)

• Negative-stain Transmission Electron Microscopy (TEM):

  • Dilute protein to approximately 0.001 mg/ml in buffer

  • Apply to glow-discharged carbon grids

  • Stain with 2% uranyl acetate

  • Image at high magnification (e.g., 180,000×)

• Circular Dichroism (CD) Spectroscopy:

  • Analysis of secondary structure composition

  • Thermal stability assessment

  • Monitoring structural changes upon ligand binding

• Thermostability Assays:

  • Utilize fluorescent dyes like CPM (7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin)

  • Monitor thermal denaturation profiles from 20°C to 95°C using fluorescence spectroscopy

These complementary approaches would provide comprehensive structural information at different resolution levels, from secondary structure composition to atomic details.

What potential functions have been proposed for UPF0232 protein Mb0004 and how can they be experimentally verified?

While the specific function of UPF0232 protein Mb0004 remains uncharacterized, several hypotheses can be proposed based on structural and sequence similarities to other proteins:

• Potential RNA metabolism role: Some UPF family proteins are involved in RNA binding and processing. The TTHA0281 protein from Thermus thermophilus, which shares structural similarities with UPF family proteins, is speculated to be involved in RNA metabolism .

• Proposed experimental approaches to verify function:

  a. RNA binding assays:

  • Electrophoretic Mobility Shift Assays (EMSA) with various RNA substrates

  • RNA immunoprecipitation followed by sequencing (RIP-seq)

  • Surface Plasmon Resonance (SPR) to determine binding affinities

  b. Enzymatic activity tests:

  • RNA cleavage assays using labeled RNA substrates

  • Phosphatase activity assays if phosphorylation sites are identified

  c. Protein interaction studies:

  • Pull-down assays to identify binding partners

  • Yeast two-hybrid screening

  • Mass spectrometry-based interactome analysis

  d. Gene knockout/knockdown studies:

  • CRISPR-Cas9 mediated gene editing in mycobacterial species

  • Analysis of phenotypic changes in growth, stress response, or pathogenicity

• Comparative genomics approach: Analyzing the conservation and genomic context of UPF0232 protein Mb0004 across different mycobacterial species could provide insights into its biological role and importance .

How can researchers investigate potential interactions between UPF0232 protein Mb0004 and other cellular components?

To investigate the potential interaction partners of UPF0232 protein Mb0004, researchers can employ the following methodologies:

• Affinity Purification coupled with Mass Spectrometry (AP-MS):

  • Express tagged UPF0232 protein Mb0004 in mycobacterial cells

  • Perform pull-down experiments using the tag

  • Identify co-purifying proteins by mass spectrometry

  • Validate interactions by co-immunoprecipitation with specific antibodies

• Surface Plasmon Resonance (SPR):

  • Immobilize purified UPF0232 protein Mb0004 on a CM7 sensor chip

  • Flow potential interaction partners over the chip

  • Measure binding kinetics and affinities

  • Typical running buffer: phosphate buffer (pH 7.4) containing 0.002% detergent to maintain protein stability

• Bacterial Two-Hybrid System:

  • Particularly useful for membrane-associated or difficult-to-express proteins

  • Screen for potential protein-protein interactions in a bacterial host

• Crosslinking Mass Spectrometry:

  • Use chemical crosslinkers to capture transient protein-protein interactions

  • Digest crosslinked complexes and analyze by mass spectrometry

  • Identify interaction interfaces at amino acid resolution

• Fluorescence Resonance Energy Transfer (FRET):

  • Tag UPF0232 protein Mb0004 and potential interaction partners with fluorescent proteins

  • Monitor protein-protein interactions in vivo

  • Provides spatial and temporal information about interactions

Each method has its strengths and limitations, so combining multiple approaches would provide the most comprehensive understanding of the protein's interaction network.

What role might UPF0232 protein Mb0004 play in mycobacterial pathogenesis and how can this be investigated?

The potential role of UPF0232 protein Mb0004 in mycobacterial pathogenesis represents an important research question, particularly given that it comes from Mycobacterium bovis, a pathogenic species closely related to M. tuberculosis. Several approaches can be used to investigate this question:

• Gene Knockout Studies:

  • Generate gene deletion mutants using specialized transduction or CRISPR-Cas9

  • Assess virulence in cellular and animal infection models

  • Compare growth kinetics under various stress conditions that mimic host environments

  • Evaluate changes in biofilm formation and drug susceptibility

• Transcriptomic and Proteomic Profiling:

  • Compare wild-type and mutant strains under various conditions

  • Identify differentially expressed genes and proteins

  • Map the regulatory networks affected by UPF0232 protein Mb0004

• Host-Pathogen Interaction Studies:

  • Investigate interactions with host immune cells

  • Analyze phagosome maturation and survival within macrophages

  • Assess cytokine responses and inflammasome activation

• Structural Vaccinology Approach:

  • Determine if UPF0232 protein Mb0004 could serve as a potential vaccine antigen

  • Analyze epitope presentation and immunogenicity

  • Evaluate protective efficacy in animal models

The insights gained from these investigations could potentially identify UPF0232 protein Mb0004 as a novel therapeutic target or biomarker for mycobacterial infections.

How can researchers differentiate between the functions of UPF0232 protein Mb0004 and other UPF family proteins in mycobacteria?

Differentiating the specific functions of UPF0232 protein Mb0004 from other UPF family proteins requires systematic comparative analysis:

• Comprehensive Sequence and Structure Analysis:

  • Multiple sequence alignment of all UPF family proteins in mycobacteria

  • Identification of conserved and variable domains

  • Structural comparison using computational modeling and experimental structures

  • Phylogenetic analysis to establish evolutionary relationships

• Parallel Functional Genomics:

  • Create a panel of knockout mutants for multiple UPF family proteins

  • Perform comparative phenotypic profiling

  • Identify unique and overlapping phenotypes

  • Conduct complementation studies to confirm specificity

• Domain Swapping Experiments:

  • Generate chimeric proteins by swapping domains between UPF0232 protein Mb0004 and other UPF proteins

  • Test functionality of chimeric proteins

  • Identify domains responsible for specific functions

• Differential Expression Analysis:

  • Compare expression patterns of different UPF proteins under various conditions

  • Identify condition-specific induction of individual UPF proteins

  • Correlate expression patterns with physiological states

• Substrate Specificity Profiling:

  • If UPF proteins have enzymatic functions, compare substrate preferences

  • Conduct competition assays between different UPF proteins

  • Identify unique substrates for UPF0232 protein Mb0004

Such comprehensive analysis would help delineate the specific role of UPF0232 protein Mb0004 within the broader context of UPF family proteins in mycobacterial physiology.

What challenges might researchers encounter when working with recombinant UPF0232 protein Mb0004 and how can these be addressed?

Researchers working with recombinant UPF0232 protein Mb0004 may encounter several technical challenges:

• Protein Solubility Issues:

  • Challenge: Recombinant proteins from mycobacteria can form inclusion bodies in heterologous expression systems.

  • Solution: Optimize expression conditions (lower temperature, reduced IPTG concentration), use solubility-enhancing tags like SUMO or MBP, or employ specialized detergents for extraction .

• Protein Stability Concerns:

  • Challenge: Maintaining stable, correctly folded protein during purification and storage.

  • Solution: Add stabilizing agents (glycerol, trehalose), optimize buffer composition, and avoid repeated freeze-thaw cycles .

  Storage Form	Recommended Conditions	Maximum Shelf Life
  Lyophilized	-20°C/-80°C	12 months
  Liquid	-20°C/-80°C with 50% glycerol	6 months
  Working aliquots	4°C	1 week

• Functional Assay Development:

  • Challenge: Establishing reliable assays without knowing the protein's function.

  • Solution: Employ unbiased screening approaches such as differential scanning fluorimetry with various ligands, or screen for interaction partners using protein arrays .

• Structural Determination Difficulties:

  • Challenge: Obtaining high-quality crystals for X-ray crystallography.

  • Solution: Screen multiple constructs with varying terminal truncations, employ surface entropy reduction mutations, or use alternative structural techniques like cryo-EM .

• Expression System Selection:

  • Challenge: Choosing between E. coli and yeast expression systems.

  • Solution: Consider parallel expression in both systems and compare protein yield, solubility, and activity. The table below summarizes key differences:

  Parameter	E. coli System	Yeast System
  Product Code	CSB-EP742566MVH-B	CSB-YP742566MVH
  Yield	Typically higher	Moderate
  Post-translational modifications	Limited	More extensive
  Folding efficiency	May require optimization	Often better for complex proteins
  Expression time	Faster (hours)	Longer (days)
  Cost	Lower	Higher

Addressing these challenges requires systematic optimization and often a combination of different approaches tailored to the specific properties of UPF0232 protein Mb0004.

How might UPF0232 protein Mb0004 interact with the nonsense-mediated mRNA decay (NMD) pathway based on knowledge of other UPF proteins?

While UPF0232 protein Mb0004 belongs to a different family than the canonical UPF1, UPF2, and UPF3 proteins involved in nonsense-mediated mRNA decay (NMD), exploring potential connections to RNA quality control pathways is worthy of investigation:

• Comparative Analysis with Canonical UPF Proteins:

  • The canonical UPF proteins (UPF1, UPF2, UPF3) are essential components of the NMD machinery that degrades mRNAs containing premature termination codons .

  • UPF0232 protein Mb0004 lacks the characteristic helicase domain of UPF1 but might still participate in RNA metabolism through different mechanisms.

• Experimental Approaches to Investigate NMD Connections:

  • RNA-protein crosslinking and immunoprecipitation followed by sequencing (CLIP-seq) to identify RNA targets

  • Tethering assays to test if UPF0232 protein Mb0004 can trigger mRNA decay when artificially bound to reporter mRNAs

  • Co-immunoprecipitation experiments to test for interactions with known NMD factors

  • Analysis of mRNA decay rates in the presence or absence of UPF0232 protein Mb0004

• Functional Complementation Tests:

  • Express UPF0232 protein Mb0004 in eukaryotic cells with UPF1/2/3 knockdowns

  • Assess if it can rescue any aspects of the NMD deficiency

  • Test if it affects translational frameshifting or nonsense codon readthrough efficiency

• Structural Basis for Potential NMD Involvement:

  • The α-β-β-β-α fold observed in related UPF family proteins could potentially mediate RNA or protein interactions relevant to RNA quality control

  • Mutation of conserved residues could help identify functional surfaces

It's important to note that evidence from studies on other UPF proteins indicates that they do not directly affect programmed frameshifting or nonsense codon readthrough in ways that are independent of their role in mRNA decay . Therefore, any potential role of UPF0232 protein Mb0004 in RNA quality control would likely involve distinct molecular mechanisms.

What techniques are recommended for assessing the purity and quality of recombinant UPF0232 protein Mb0004 preparations?

Ensuring high purity and quality of recombinant UPF0232 protein Mb0004 is critical for reliable research outcomes. The following analytical techniques are recommended:

• Gel Electrophoresis:

  • SDS-PAGE with Coomassie Blue staining for basic purity assessment (>85% purity standard)

  • Both reducing and non-reducing conditions should be tested to evaluate potential disulfide bond formation

  • Western blotting with anti-His tag antibodies to confirm identity

• Mass Spectrometry:

  • LC-MS/MS for accurate molecular weight determination and confirmation of sequence coverage

  • Intact protein mass analysis to detect potential post-translational modifications

  • Peptide mass fingerprinting after tryptic digestion for protein identification

• Size Exclusion Chromatography (SEC):

  • Analytical SEC to assess homogeneity and oligomeric state

  • Multi-angle light scattering (SEC-MALS) for precise molecular weight determination in solution

  • Recommended column: Superdex 200 Increase 10/300

• Dynamic Light Scattering (DLS):

  • Measurement of size distribution to detect aggregation

  • Determination of hydrodynamic radius

  • Assessment of sample polydispersity

• Circular Dichroism (CD) Spectroscopy:

  • Evaluation of secondary structure content

  • Thermal stability assessment

  • Monitoring of proper folding

• Thermal Shift Assays:

  • Using fluorescent dyes like SYPRO Orange or CPM

  • Determination of melting temperature (Tm) as quality control parameter

  • Screening of buffer conditions for optimal stability

A typical quality control workflow would include SDS-PAGE analysis (minimum 85% purity), SEC analysis for homogeneity assessment, and at least one spectroscopic method to confirm proper folding.

What experimental design considerations are important when comparing UPF0232 protein Mb0004 from different expression systems?

When comparing UPF0232 protein Mb0004 produced in different expression systems (e.g., E. coli vs. yeast), the following experimental design considerations are crucial for valid comparisons:

• Construct Design Standardization:

  • Use identical amino acid sequences including tags

  • Maintain consistent tag position (N- or C-terminal)

  • Consider codon optimization specific to each expression host

  • Document the specific vector elements (promoters, terminators) used

• Expression and Purification Controls:

  • Implement parallel purification protocols where possible

  • When system-specific protocols are required, include control proteins expressed in both systems

  • Normalize protein concentrations accurately for all comparative assays

  • Document lot-to-lot variation within each system

• Analytical Comparisons:

  • Conduct side-by-side analysis on the same gel/chromatography run

  • Include internal standards for quantitative comparisons

  • Utilize multiple complementary techniques (e.g., CD spectroscopy, thermal shift assays)

• Functional Assay Design:

  • Develop quantitative activity assays

  • Test across multiple substrate concentrations

  • Include appropriate positive and negative controls

  • Calculate and compare kinetic parameters rather than single-point measurements

• Systematic Data Collection:

  Parameter	E. coli-expressed	Yeast-expressed	Measurement Method
  Yield (mg/L culture)			Bradford/BCA assay
  Purity (%)			Densitometry of SDS-PAGE
  Activity (units/mg)			Specific activity assay
  Molecular weight			Mass spectrometry
  Thermal stability (Tm)			DSF/CD thermal melt
  Secondary structure			Circular dichroism
  Oligomeric state			SEC-MALS
  Glycosylation status			Glycoprotein staining

• Statistical Analysis:

  • Perform experiments with at least three biological replicates

  • Apply appropriate statistical tests (t-test, ANOVA)

  • Report effect sizes alongside p-values

  • Consider Bayesian approaches for complex comparisons