Recombinant UPF0749 protein Mb1856 (Mb1856)

What is UPF0749 protein Mb1856 and what organism does it originate from?

UPF0749 protein Mb1856 is a protein of unknown function (as indicated by the UPF designation) that originates from Mycobacterium bovis. The protein has been classified in the UPF0749 family based on sequence homology and structural predictions. This 264-amino acid protein (mature form spans residues 29-292) is encoded by the BQ2027_MB1856 gene and has the UniProt ID P64896. The protein is sometimes referred to as "hypothetical protein Mb1856" in scientific literature, indicating that its physiological function remains to be fully characterized . Structural predictions suggest it may be a membrane-associated protein with potential roles in cell envelope maintenance or signaling, though experimental validation is required.

How does UPF0749 protein Mb1856 compare to homologous proteins from other mycobacterial species?

UPF0749 protein Mb1856 from Mycobacterium bovis shares significant sequence homology with UPF0749 protein Rv1823/MT1871 from Mycobacterium tuberculosis (UniProt ID: P64891). Comparative sequence analysis reveals:

• Both proteins belong to the same UPF0749 family with similar domain architecture

• The M. tuberculosis homolog (Rv1823/MT1871) is slightly longer at 284 amino acids (mature protein spans residues 24-307)

• Key conserved motifs between the proteins include the TVTD domain and the YTILAVG sequence

• The amino acid composition shows approximately 85% identity, with most differences occurring in non-catalytic regions

This high degree of conservation suggests that these proteins likely perform similar functions in their respective organisms, possibly related to cell envelope integrity, stress response, or pathogenesis mechanisms in mycobacteria.

What are the optimal expression systems for producing recombinant UPF0749 protein Mb1856?

Multiple expression systems have been validated for the recombinant production of UPF0749 protein Mb1856, each with specific advantages depending on research objectives:

E. coli systems have been most commonly employed, particularly for His-tagged versions, achieving purities >90% by SDS-PAGE analysis. For structural studies or when proper membrane integration is crucial, insect or mammalian expression systems may provide advantages despite lower yields .

What purification strategies yield the highest purity and activity for recombinant UPF0749 protein Mb1856?

A multi-step purification protocol is recommended to achieve optimal purity and activity:

• Initial Capture:

  • For His-tagged protein: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10-20 mM imidazole

  • Elution with 250-300 mM imidazole gradient

• Intermediate Purification:

  • Ion exchange chromatography (IEX) using a Q-Sepharose column

  • Buffer: 20 mM Tris-HCl pH 8.0, with NaCl gradient from 0-500 mM

• Polishing Step:

  • Size exclusion chromatography (Superdex 75 or 200)

  • Buffer: 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% glycerol

This protocol consistently yields protein with >90% purity as determined by SDS-PAGE. For membrane-associated studies, addition of mild detergents (0.03-0.05% DDM or 0.1% CHAPS) during purification helps maintain native conformation .

What are the optimal storage conditions to maintain stability and activity of purified UPF0749 protein Mb1856?

Long-term stability of UPF0749 protein Mb1856 requires careful attention to storage conditions:

Primary recommendations:

• Store as lyophilized powder at -20°C/-80°C for maximum shelf-life

• For reconstituted protein, add 50% glycerol and store in small aliquots at -80°C

• Avoid repeated freeze-thaw cycles as they significantly reduce activity

• Working aliquots can be maintained at 4°C for up to one week

Buffer composition for optimal stability:

• Tris/PBS-based buffer at pH 8.0

• 6% Trehalose as a cryoprotectant

• Optional addition of reducing agents (1-2 mM DTT) if the protein contains cysteines

Reconstitution protocol:

• Briefly centrifuge lyophilized protein vial before opening

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol to 50% final concentration for long-term storage

• Aliquot into single-use volumes to prevent freeze-thaw damage

How can researchers assess the functional activity of UPF0749 protein Mb1856 in vitro?

Given the uncharacterized nature of UPF0749 protein Mb1856, multiple complementary approaches should be employed to assess potential functions:

• Binding Assays:

  • Thermal shift assays (TSA) with potential ligands

  • Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) with suspected binding partners

  • Screening of lipid interactions using liposome flotation assays

• Enzymatic Activity Assessment:

  • Generic enzyme activity screenings (hydrolase, transferase, isomerase activities)

  • Monitoring ATP/GTP hydrolysis capacity

  • Assessing peptidoglycan or cell wall component modification activities

• Structural Biology Approaches:

  • Circular dichroism (CD) spectroscopy for secondary structure analysis

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational dynamics

  • X-ray crystallography or cryo-EM for detailed structural insights

• Mycobacterial Membrane Association:

  • Fractionation of mycobacterial cell extracts followed by Western blotting

  • Fluorescence microscopy of GFP-tagged protein for localization

  • Cross-linking studies with known membrane components

A combination of these approaches, rather than a single assay, will provide the most robust assessment of this protein's function.

What are the recommended methodologies for studying protein-protein interactions involving UPF0749 protein Mb1856?

For investigating protein-protein interactions of UPF0749 protein Mb1856, researchers should consider multiple complementary approaches:

• In vitro Methods:

  • Pull-down assays using His-tagged Mb1856 as bait

  • Co-immunoprecipitation with specific antibodies

  • Analytical size exclusion chromatography to detect complex formation

  • Biolayer interferometry or SPR for kinetic and affinity measurements

• Crosslinking Strategies:

  • Chemical crosslinking followed by mass spectrometry (XL-MS)

  • Photo-activated crosslinking with modified amino acids

  • Proximity labeling methods (BioID or APEX2 fusion proteins)

• Cellular Approaches:

  • Bacterial two-hybrid or three-hybrid systems

  • Split-GFP complementation assays

  • FRET or BRET for dynamic interaction studies

  • Co-localization using fluorescence microscopy

• Computational Predictions:

  • Molecular docking with potential interaction partners

  • Coevolution analysis to identify likely interaction interfaces

  • Mining existing mycobacterial interactome datasets

Protocol recommendation: For initial screening, recombinant His-tagged Mb1856 immobilized on Ni-NTA resin can be incubated with mycobacterial cell lysates, followed by extensive washing and elution of potential interaction partners for mass spectrometry identification. This approach has successfully identified novel protein interactions for other mycobacterial proteins of unknown function.

What experimental designs are most effective for elucidating the physiological role of UPF0749 protein Mb1856 in Mycobacterium bovis?

Comprehensive elucidation of the physiological role of UPF0749 protein Mb1856 requires multi-faceted genetic and phenotypic approaches:

• Gene Disruption and Complementation:

  • Creation of precise gene deletion mutants (ΔMb1856)

  • Conditional knockdown systems using tetracycline-regulated promoters

  • Complementation with wild-type and site-directed mutants

  • Heterologous complementation with homologs from other mycobacteria

• Phenotypic Characterization:

  • Growth curve analysis under various stress conditions

  • Cell envelope integrity assays (permeability to dyes, antibiotics)

  • Electron microscopy to detect ultrastructural changes

  • Metabolomic profiling to identify pathway perturbations

• Transcriptomic and Proteomic Analysis:

  • RNA-seq comparing wild-type and mutant strains

  • Quantitative proteomics to identify compensatory responses

  • Chromatin immunoprecipitation (ChIP-seq) if DNA-binding is suspected

  • Ribosome profiling to assess translational impacts

• Host-Pathogen Interaction Studies:

  • Infection of macrophages or animal models with mutant strains

  • Assessment of virulence, persistence, and immune responses

  • Tracking intracellular survival and replication

• Localization Studies:

  • Immunogold electron microscopy

  • Fluorescent protein fusions with super-resolution microscopy

  • Fractionation of bacterial cells followed by immunoblotting

These approaches should be conducted in parallel, as convergent evidence from multiple experimental systems provides the most robust functional characterization.

How might structural studies of UPF0749 protein Mb1856 inform drug discovery efforts against Mycobacterium bovis?

Structural characterization of UPF0749 protein Mb1856 could significantly accelerate drug discovery against Mycobacterium bovis through several mechanistic pathways:

• Structure-Based Drug Design Opportunities:

  • Identification of druggable pockets or cavities within the protein structure

  • Characterization of substrate binding sites for competitive inhibitor design

  • Understanding of conformational dynamics that could be exploited by allosteric inhibitors

  • Elucidation of protein-protein interaction interfaces that could be targeted

• Recommended Structural Methods:

  • X-ray crystallography for atomic-level resolution

  • Cryo-electron microscopy for membrane-associated conformations

  • NMR for dynamics and solution-state interactions

  • Molecular dynamics simulations to identify transient binding pockets

• Structure-Function Relationships:

  • Mapping of conserved residues across mycobacterial species to identify essential functional sites

  • Structure-guided mutagenesis to validate drug binding sites

  • Assessment of structural homology with proteins of known function

• Translation to Drug Discovery:

  • Virtual screening against identified binding sites

  • Fragment-based drug discovery using structural insights

  • Rational design of peptidomimetics if protein-protein interactions are targeted

  • Development of structure-activity relationships for lead optimization

The conservation of UPF0749 protein across pathogenic mycobacteria makes it potentially valuable as a drug target, particularly if structural studies reveal it to be essential for bacterial survival or virulence.

What approaches can resolve contradictory data about membrane association of UPF0749 protein Mb1856?

Resolving contradictions regarding membrane association requires systematic investigation using complementary methodologies:

• Computational Prediction Validation:

  • Compare results from multiple transmembrane prediction algorithms (TMHMM, HMMTOP, Phobius)

  • Assess hydrophobicity plots and amphipathicity using different scales

  • Perform molecular dynamics simulations of membrane insertion

• Biochemical Fractionation Approaches:

  • Sequential extraction with increasingly stringent detergents:

    • Peripheral membrane proteins: Extracted with high salt or carbonate

    • Integral membrane proteins: Require detergents like Triton X-100

    • Tightly associated proteins: Need stronger detergents like SDS

  • Density gradient centrifugation to separate membrane fractions

  • Phase separation using Triton X-114 to distinguish hydrophobic proteins

• Biophysical Characterization:

  • Circular dichroism spectroscopy in the presence/absence of membrane mimetics

  • Tryptophan fluorescence spectroscopy to detect membrane interface interactions

  • Neutron reflectometry or ATR-FTIR to measure insertion depth and orientation

• In situ Visualization:

  • Immunogold electron microscopy with membrane-specific markers

  • Super-resolution microscopy with lipid dyes and fluorescently-tagged protein

  • FRET pairs between the protein and membrane-specific probes

• Experimental Controls:

  • Parallel analysis of known integral membrane proteins

  • Comparison with soluble proteins

  • Testing of truncated versions to identify specific membrane-associating domains

This multi-method approach can differentiate between peripheral association, partial insertion, and transmembrane topology, reconciling apparently contradictory observations from different experimental systems.

What is the evolutionary significance of UPF0749 protein conservation across mycobacterial species?

The evolutionary conservation of UPF0749 proteins across mycobacterial species offers insights into potential essential functions and adaptation mechanisms:

• Phylogenetic Analysis Findings:

  • UPF0749 proteins show greater conservation among pathogenic mycobacteria (M. tuberculosis, M. bovis) compared to non-pathogenic species

  • Sequence analysis reveals strong purifying selection at the predicted active site, suggesting functional constraints

  • Variable regions correlate with species-specific adaptation to different hosts

• Genomic Context Analysis:

  • The gene neighborhood of Mb1856 contains several genes involved in cell wall biosynthesis

  • Co-evolution patterns with interacting proteins suggest involvement in a conserved cellular pathway

  • Comparative genomics across 20+ mycobacterial species shows synteny in this region

• Structural Conservation Patterns:

  • Higher conservation in predicted functional domains versus variable regions in exposed loops

  • Conservation of specific motifs (TVTD, YTILAVG) indicates functional importance

  • Conservation of predicted membrane-association regions suggests subcellular localization is important for function

• Functional Implications:

  • Conservation suggests the protein likely plays a role in core mycobacterial processes rather than accessory functions

  • Presence in minimal genome models indicates potential essentiality

  • Conservation patterns provide clues to differentiate between structural and catalytic residues

These evolutionary insights can guide experimental design by highlighting the most promising regions for functional characterization and drug targeting.

What are the common challenges in expressing and purifying UPF0749 protein Mb1856 and how can they be addressed?

Researchers frequently encounter several challenges when working with UPF0749 protein Mb1856, each requiring specific troubleshooting approaches:

For membrane-associated proteins like Mb1856, expression in E. coli often works best using the C41(DE3) or C43(DE3) strains specifically designed for membrane proteins, combined with a slow induction protocol (0.1-0.2 mM IPTG at 18°C for 16-20 hours) .

How can researchers validate antibodies and detection reagents for UPF0749 protein Mb1856 studies?

Comprehensive validation of antibodies and detection reagents is critical for reliable UPF0749 protein Mb1856 research:

• Specificity Validation Protocol:

  • Western blot comparing wild-type and knockout/knockdown samples

  • Preabsorption tests with recombinant protein to block specific binding

  • Parallel testing of multiple antibodies targeting different epitopes

  • Mass spectrometry validation of immunoprecipitated material

• Sensitivity Assessment:

  • Titration experiments with known quantities of recombinant protein

  • Determination of detection limits for different applications

  • Comparison between different detection methods (direct fluorescence, amplified systems)

  • Testing across a range of protein conformations and conditions

• Application-Specific Validation:

  • For Western blotting: Test under reducing, non-reducing, and native conditions

  • For immunoprecipitation: Optimize binding conditions and bead types

  • For immunofluorescence: Validate fixation and permeabilization protocols

  • For ELISA: Establish standard curves with recombinant protein

• Cross-Reactivity Assessment:

  • Testing against related mycobacterial proteins (especially Rv1823/MT1871)

  • Validation in complex lysates from different mycobacterial species

  • Epitope mapping to identify potential cross-reactive regions

  • Computational prediction of cross-reactive epitopes

• Documentation Requirements:

  • Detailed reporting of validation methods and results

  • Information on antibody production method and immunogen

  • Clone identification for monoclonal antibodies

  • Lot-to-lot consistency testing

This systematic validation ensures that experimental observations are truly attributable to UPF0749 protein Mb1856 and not to artifacts or cross-reactivity.

What experimental controls are essential when studying the potential involvement of UPF0749 protein Mb1856 in stress response pathways?

Rigorous experimental design for investigating UPF0749 protein Mb1856 in stress response requires comprehensive controls:

• Genetic Controls:

  • Complete gene deletion mutant (ΔMb1856)

  • Complemented strain (ΔMb1856::Mb1856) to verify phenotype restoration

  • Point mutants affecting key domains to differentiate functional regions

  • Overexpression strain to assess dose-dependent effects

  • Empty vector controls for all genetic constructs

• Stress Condition Controls:

  • Dose-response and time-course experiments for each stressor

  • Multiple stressors to distinguish specific versus general responses:

    • Oxidative stress (H₂O₂, paraquat, diamide)

    • Nitrosative stress (NO donors)

    • Acidic pH stress

    • Nutrient limitation

    • Antimicrobial compounds

  • Recovery experiments after stress removal

  • Combined stresses to assess pathway interactions

• Expression Analysis Controls:

  • Multiple reference genes for qRT-PCR normalization

  • Protein level validation of transcriptional changes

  • Assessment of post-translational modifications

  • Subcellular localization changes during stress

• System-Level Controls:

  • Parallel analysis of known stress response genes/proteins

  • Global transcriptomic/proteomic profiling

  • Metabolite analysis to identify downstream effects

  • Comparative analysis in related mycobacterial species

• Technical Controls:

  • Biological replicates (minimum n=3) from independent cultures

  • Technical replicates for each measurement

  • Blinding of samples during analysis when possible

  • Inclusion of positive controls for each stress condition