Recombinant Uncharacterized protein Mb1843c (Mb1843c)

What is known about the basic structure of Mb1843c protein?

Mb1843c is an uncharacterized protein from Mycobacterium bovis with 143 amino acids. The full amino acid sequence is: "MITNLRRRTAMAAAGLGAALGLGILLVPTVDAHLANGSMSEVMMSEIAGLPIPPIIHYGA IAYAPSGASGKAWHQRTPARAEQVALEKCGDKTCKVVSRFTRCGAVAYNGSKYQGGTGLT RRAAEDDAVNRLEGGRIVNWACN" . Initial sequence analysis suggests it contains hydrophobic regions that may indicate membrane association, particularly in the N-terminal region. Current structural data remains limited, making this protein an excellent candidate for fundamental characterization studies. Researchers should consider combining computational structure prediction with experimental validation through circular dichroism spectroscopy or X-ray crystallography.

What expression systems are available for producing recombinant Mb1843c?

Multiple expression systems have been developed for recombinant Mb1843c production, each with distinct advantages for different research applications. The most common systems include:

E. coli remains the most commonly used system, yielding full-length protein (1-143aa) fused to an N-terminal His tag . When selecting an expression system, researchers should consider downstream applications and whether post-translational modifications are critical to their study objectives.

What are the optimal conditions for storing and reconstituting recombinant Mb1843c?

For long-term preservation of Mb1843c activity, store the lyophilized protein at -20°C to -80°C . Aliquoting is necessary to prevent degradation from repeated freeze-thaw cycles. For reconstitution, centrifuge the vial briefly prior to opening, then reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL . Adding glycerol to a final concentration of 5-50% (with 50% being standard) is recommended for long-term storage . Working aliquots may be stored at 4°C for up to one week, but repeated freeze-thaw cycles will compromise protein integrity. The storage buffer typically consists of Tris/PBS-based buffer with 6% Trehalose at pH 8.0 .

What purification strategies yield the highest purity of recombinant Mb1843c?

Given that commercially available Mb1843c comes with an N-terminal His-tag, immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co2+ resins represents the primary purification strategy. To achieve research-grade purity (>90% as assessed by SDS-PAGE) , a multi-step purification protocol is recommended:

• Initial IMAC purification using standard imidazole elution gradients

• Intermediate ion-exchange chromatography to separate charged contaminants

• Final size-exclusion chromatography to achieve highest purity

For specialized applications requiring ultra-high purity, consider additional purification steps such as hydroxyapatite chromatography or hydrophobic interaction chromatography, especially if membrane-association properties of Mb1843c impact standard purification efficacy.

What bioinformatic approaches can predict potential functions of Mb1843c?

When working with uncharacterized proteins like Mb1843c, integrating multiple bioinformatic approaches can provide valuable insights into potential functions:

• Sequence homology analysis: Compare Mb1843c with characterized proteins across species using BLAST, HMMER, and PSI-BLAST to identify distant homologs.

• Domain prediction: Tools like PFAM, SMART, and InterProScan can identify conserved domains that suggest function.

• Secondary structure prediction: PSIPRED and JPred can reveal structural motifs indicative of specific functions.

• Subcellular localization prediction: Tools like TMHMM, SignalP, and PSORT can predict cellular localization, particularly relevant given the hydrophobic regions in Mb1843c.

• Protein-protein interaction prediction: STRING and STITCH databases may reveal potential binding partners.

The hydrophobic N-terminal region of Mb1843c suggests possible membrane association, which should be factored into functional predictions and experimental design. Cross-referencing predictions from multiple tools will provide the most reliable foundation for experimental validation.

What spectroscopic methods are most suitable for analyzing Mb1843c secondary structure?

For a comprehensive analysis of Mb1843c secondary structure, employ a multi-method approach similar to that used for other uncharacterized proteins:

• Circular Dichroism (CD) Spectroscopy: The most accessible method for estimating secondary structure content (α-helices, β-sheets, random coils). Based on approaches used with similar proteins, a wavelength scan from 190-260 nm in a 0.1 cm path length cell with protein concentration at 0.1-0.2 mg/mL would be appropriate .

• Fourier Transform Infrared (FTIR) Spectroscopy: Complements CD by providing additional information about β-sheet structures that may be challenging to resolve with CD alone.

• Nuclear Magnetic Resonance (NMR) Spectroscopy: For detailed atomic-level structural information, though this requires isotopically labeled protein samples.

• X-ray Crystallography: The gold standard for high-resolution structural determination if Mb1843c can be successfully crystallized.

When interpreting spectroscopic data for Mb1843c, compare with data from proteins of known structure to estimate secondary structure content percentages. Learning from approaches used with UNC-18 protein characterization, where alpha-helix and beta-sheet content were determined to be 10.0% and 59.0% respectively using CD spectroscopy , could provide methodological guidance.

How can protein-protein interaction studies be designed to identify Mb1843c binding partners?

To systematically identify binding partners of the uncharacterized Mb1843c protein, employ a multi-faceted approach:

• Pull-down assays: Leverage the His-tag on recombinant Mb1843c for affinity purification of protein complexes from Mycobacterium bovis lysates. Analyze bound proteins using mass spectrometry.

• Yeast two-hybrid (Y2H) screening: Create a fusion of Mb1843c with a DNA-binding domain and screen against a library of Mycobacterium proteins fused to activation domains.

• Surface Plasmon Resonance (SPR): Immobilize purified Mb1843c on sensor chips to quantitatively measure binding kinetics with candidate interacting proteins.

• Co-immunoprecipitation (Co-IP): Generate specific antibodies against Mb1843c for precipitation of native protein complexes from bacterial lysates.

• Bacterial two-hybrid systems: Particularly useful for membrane-associated proteins like Mb1843c with hydrophobic regions.

When interpreting results, prioritize interactions identified across multiple methods. This approach parallels successful methods used to characterize interactions between other uncharacterized proteins, such as UNC-18's binding to syntaxin , which provided crucial functional insights.

What are the best methods for generating antibodies against Mb1843c for research applications?

Developing specific antibodies against Mb1843c requires careful consideration of epitope selection and immunization strategies:

• Epitope selection: Analyze the Mb1843c sequence for highly antigenic regions using prediction algorithms (Bepipred, ABCpred). Avoid highly hydrophobic regions that may be inaccessible in the native protein.

• Polyclonal antibody production:

  • Use purified full-length recombinant Mb1843c (>90% purity) for immunization

  • Employ a standard 8-12 week immunization protocol with adjuvants

  • Test antibody specificity against both recombinant protein and native protein in Mycobacterium bovis extracts

• Monoclonal antibody production:

  • Consider using synthetic peptides from predicted exposed epitopes conjugated to carrier proteins

  • Screen hybridomas against both peptide and full-length protein

  • Validate specificity in immunoblotting, immunoprecipitation, and immunofluorescence applications

• Validation controls:

  • Pre-immune serum controls

  • Absorption controls with recombinant antigen

  • Cross-reactivity testing against related mycobacterial proteins

For specialized applications like structural studies, consider generating Fab fragments or single-chain variable fragments (scFvs) that may offer advantages in co-crystallization experiments.

What approaches can determine if Mb1843c has enzymatic activity?

Investigating potential enzymatic functions of uncharacterized proteins like Mb1843c requires systematic screening approaches:

• Sequence-based predictions: Scan for catalytic motifs using tools like PROSITE, PRINTS, and the Conserved Domain Database.

• Activity-based screening panel: Test purified Mb1843c against a diverse panel of standard substrates for common enzymatic activities:

  • Hydrolase activity (esterase, phosphatase, glycosidase)

  • Transferase activity (methyltransferase, acyltransferase)

  • Oxidoreductase activity

  • Lyase activity

• Substrate and cofactor requirements: Systematically test activity in the presence of potential cofactors (metal ions, nucleotides) and under varying pH and temperature conditions.

• Targeted assays based on localization: Given the potential membrane association of Mb1843c, focus on activities relevant to membrane proteins (transporters, channels, signaling proteins).

• Metabolomics approach: Compare metabolite profiles of bacterial systems with and without Mb1843c expression to identify potential substrates or products.

Document negative results thoroughly, as establishing what activities Mb1843c does not possess is equally valuable for narrowing the functional search space.

How can researchers evaluate the potential role of Mb1843c in Mycobacterium bovis pathogenesis?

To investigate Mb1843c's potential role in pathogenesis, implement a comprehensive experimental strategy:

• Gene knockout/knockdown studies:

  • Create Mb1843c deletion mutants in Mycobacterium bovis

  • Assess phenotypic changes in growth, survival, and virulence

• Expression analysis:

  • Quantify Mb1843c expression during different growth phases

  • Compare expression levels between virulent and attenuated strains

  • Analyze expression during host cell infection using qRT-PCR

• Host-pathogen interaction studies:

  • Assess impact of Mb1843c knockout on survival within macrophages

  • Evaluate inflammatory responses in infected host cells

  • Determine contribution to granuloma formation in appropriate models

• Localization studies:

  • Determine subcellular localization using fluorescent protein fusions or immunogold electron microscopy

  • Assess whether Mb1843c is secreted or surface-exposed during infection

• Animal infection models:

  • Compare wild-type and Mb1843c mutant strains in appropriate animal models

  • Evaluate bacterial load, tissue damage, and immune responses

This multi-faceted approach can reveal whether Mb1843c contributes to virulence, potentially identifying it as a target for therapeutic interventions against Mycobacterium bovis infections.

What strategies can overcome solubility issues when working with recombinant Mb1843c?

The hydrophobic regions in Mb1843c may present solubility challenges during expression and purification. Implement these strategies to improve solubility:

• Expression optimization:

  • Test multiple expression temperatures (16°C, 25°C, 37°C)

  • Use specialized E. coli strains designed for membrane proteins (C41, C43)

  • Co-express with chaperones (GroEL/GroES, DnaK/DnaJ)

• Buffer optimization:

  • Screen different pH conditions (range 6.0-9.0)

  • Test various salt concentrations (100-500 mM NaCl)

  • Include solubility enhancers: glycerol (5-20%), non-detergent sulfobetaines, arginine

• Detergent screening:

  • Mild non-ionic detergents (DDM, LDAO, OG)

  • Zwitterionic detergents (CHAPS, Fos-Choline)

  • Systematic testing with detergent screening kits

• Alternative solubilization methods:

  • Nanodiscs for maintaining native membrane environment

  • Amphipols for stabilizing membrane proteins in aqueous solution

  • SMALPs (styrene maleic acid lipid particles) for extraction with native lipids

When reconstituting lyophilized Mb1843c, follow the recommended protocol using deionized sterile water to a concentration of 0.1-1.0 mg/mL, and consider adding glycerol to enhance stability . For highest solubility, maintain the protein in Tris/PBS-based buffer with 6% Trehalose at pH 8.0 .

How can researchers troubleshoot low yields in recombinant Mb1843c expression?

When facing low expression yields of Mb1843c, systematically address potential issues at each production stage:

• Expression vector optimization:

  • Optimize codon usage for the expression host

  • Test different promoter strengths (T7, tac, araBAD)

  • Evaluate the impact of different fusion tags (His, GST, MBP, SUMO)

• Expression conditions:

  • Media composition (LB, TB, autoinduction media)

  • IPTG concentration titration (0.1-1.0 mM)

  • Duration of induction (2-24 hours)

  • Cell density at induction (OD600 0.4-1.0)

• Cell lysis troubleshooting:

  • Compare mechanical (sonication, French press) and chemical lysis methods

  • Optimize lysis buffer composition to prevent aggregation

  • Include appropriate protease inhibitors to prevent degradation

• Expression toxicity mitigation:

  • Use tight expression control with glucose repression

  • Test bacterial strains with reduced protease activity

  • Consider expression in cell-free systems for highly toxic proteins

• Recovery optimization:

  • Optimize centrifugation speeds and times for membrane fractions

  • Test different solubilization conditions if protein is membrane-associated

  • Implement affinity purification strategies that maximize recovery

Document all optimization attempts systematically in a laboratory notebook, recording detailed conditions and corresponding yields to identify the most critical parameters affecting Mb1843c production.

What are promising approaches for determining the physiological role of Mb1843c in Mycobacterium bovis?

Elucidating the physiological role of Mb1843c requires innovative approaches combining genetics, biochemistry, and systems biology:

• Conditional expression systems:

  • Develop regulatable expression systems to study the effects of Mb1843c depletion or overexpression

  • Use CRISPRi for tunable knockdown to identify phenotypes associated with different expression levels

• Interactome mapping:

  • Perform comprehensive protein-protein interaction studies using proximity labeling (BioID, APEX)

  • Identify genetic interactions through synthetic lethal screens or Tn-Seq in relevant stress conditions

• Transcriptional response analysis:

  • RNA-Seq comparing wild-type and Mb1843c mutant strains under various conditions

  • ChIP-Seq if Mb1843c shows any DNA-binding potential

• Metabolomic and lipidomic profiling:

  • Assess changes in metabolite and lipid profiles in Mb1843c mutants

  • Focus particularly on membrane composition given the potential membrane association of Mb1843c

• Comparative genomics approach:

  • Analyze the conservation and synteny of Mb1843c across mycobacterial species

  • Correlate presence/absence or sequence variation with specific phenotypes or niches

Integrating data across these methods will build a comprehensive understanding of Mb1843c's role in mycobacterial physiology and potentially reveal whether it represents a viable target for therapeutic intervention.

How might structural biology approaches contribute to understanding Mb1843c function?

Structural biology will be crucial for elucidating Mb1843c function, especially given its uncharacterized status. Consider these advanced approaches:

• X-ray crystallography:

  • Screen crystallization conditions systematically

  • Consider crystallization with potential binding partners

  • Use molecular replacement with homologous structures if identified

• Cryo-electron microscopy:

  • Particularly valuable if Mb1843c forms complexes or associates with membranes

  • Single-particle analysis for soluble forms

  • Electron tomography for membrane-associated states

• NMR spectroscopy:

  • Solution NMR for dynamic regions and ligand binding

  • Solid-state NMR for membrane-associated domains

  • Study dynamics using relaxation measurements

• Integrative structural biology:

  • Combine low-resolution techniques (SAXS, SANS) with high-resolution methods

  • Use cross-linking mass spectrometry (XL-MS) to identify domain interactions

  • Employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map functional regions

• Computational structure prediction:

  • Apply AlphaFold2 and RoseTTAFold for initial structural models

  • Refine predictions with experimental constraints

  • Use molecular dynamics simulations to explore conformational flexibility

Following successful structural determination, structure-guided mutagenesis can validate functional hypotheses by creating targeted mutations in predicted active sites or interaction interfaces. This approach has proven valuable for other initially uncharacterized bacterial proteins.

How does Mb1843c compare with homologous proteins in other mycobacterial species?

While Mb1843c remains uncharacterized, comparative analysis with homologous proteins can provide functional insights:

• Sequence conservation analysis:

  • Compare Mb1843c with homologs in M. tuberculosis, M. avium, and other mycobacteria

  • Identify conserved residues that may indicate functional importance

  • Construct phylogenetic trees to understand evolutionary relationships

• Genomic context comparison:

  • Analyze genes neighboring Mb1843c and its homologs across species

  • Identify conserved gene clusters that may suggest functional associations

  • Look for co-evolution patterns with other proteins

• Expression pattern comparison:

  • Compare expression profiles of Mb1843c homologs under various conditions

  • Identify common regulatory patterns across species

  • Correlate expression with specific physiological states or stress responses

Such comparative approaches may reveal whether Mb1843c represents a core mycobacterial function or a species-specific adaptation, guiding further experimental design for functional characterization.

What can researchers learn from studying other uncharacterized proteins that were successfully characterized?

The characterization journey of previously uncharacterized proteins offers valuable methodological insights for Mb1843c research:

• Case studies of successful characterization:

  • UNC-18 protein began as uncharacterized but was eventually shown to regulate vesicle trafficking through interaction with syntaxin

  • SANBR (KIAA1841) was initially uncharacterized but later found to function as a negative regulator in B cell processes

• Methodology evolution:

  • Integration of structural, biochemical, and genetic approaches proved most successful

  • Identification of binding partners often provided the first functional clues

  • Expression pattern analysis in different conditions revealed physiological contexts

• Common technical hurdles:

  • Solving expression and purification challenges

  • Developing specific antibodies and validation tools

  • Establishing physiologically relevant assay systems