Recombinant Mycobacterium avium UPF0233 membrane protein MAV_0015 (MAV_0015)

What is MAV_0015 and what is its genomic context in Mycobacterium avium?

MAV_0015 is a UPF0233 membrane protein from Mycobacterium avium, also known as Cell division protein CrgA. It is encoded by the crgA gene located at positions 14814-15095 on the negative strand of the Mycobacterium avium 104 complete genome. The gene is 282 base pairs long and encodes a protein of 94 amino acids. MAV_0015 belongs to Orthogroup Number 11 and has orthologous genes in several other mycobacterial species .

What is the amino acid sequence of the MAV_0015 protein?

The full amino acid sequence of MAV_0015 protein (residues 1-93) is:
MPKSKVRKKNDFTVSAVSRTPVKVKVGPSSVWFVALFIGLMLIGLVWLMVFQLAAVGSQAPTALNWMAQLGPWNYAIAFAFMITGLLLTMRWH

What structural features characterize the MAV_0015 protein?

MAV_0015 is classified as a membrane protein, suggesting it contains hydrophobic regions that anchor it within the cell membrane. Analysis of its amino acid sequence reveals hydrophobic stretches consistent with transmembrane domains. As a UPF0233 family protein, it shares conserved structural features with other members of this family, though the specific three-dimensional structure of MAV_0015 has not been fully characterized in the provided literature .

What is the relationship between MAV_0015 (crgA) and Mycobacterium avium infections?

Mycobacterium avium complex (MAC) infections are increasingly common worldwide and can be difficult to treat, often requiring a year or more of antibiotic therapy. While the specific role of MAV_0015 in pathogenesis is not directly addressed in the search results, as a cell division protein, it likely plays a role in bacterial replication and survival. Understanding proteins involved in essential cellular processes like cell division could potentially reveal new therapeutic targets for treating MAC infections, which have a mortality rate of approximately 25% within 5 years .

How does the function of MAV_0015 compare to its orthologs in other mycobacterial species?

MAV_0015 has several orthologs in other mycobacterial species, including Rv0011c in Mycobacterium tuberculosis, MAP0013c in Mycobacterium avium subsp. paratuberculosis, and ML0013 in Mycobacterium leprae. Comparative functional analysis of these orthologs can provide insights into conserved biological roles across pathogenic mycobacteria. Research methodologies should include:

• Sequence alignment of all orthologs to identify conserved domains

• Structural prediction using homology modeling

• Gene knockout studies in multiple species to compare phenotypic effects

• Complementation experiments to test functional conservation

• Protein-protein interaction studies to map conserved interactomes

What experimental approaches are most effective for studying the membrane topology of MAV_0015?

To elucidate the membrane topology of MAV_0015, researchers should consider a multi-method approach:

• Computational prediction: Use algorithms like TMHMM, Phobius, or TOPCONS to predict transmembrane regions

• Cysteine scanning mutagenesis: Introduce cysteine residues at various positions and determine their accessibility to membrane-impermeable reagents

• Protein fusion techniques: Create fusions with reporter proteins (e.g., GFP, PhoA) at different positions to determine cytoplasmic versus periplasmic localization

• Protease protection assays: Determine which regions are protected from proteolytic digestion by the membrane

• Cryo-electron microscopy: For high-resolution structural analysis in native-like membrane environments

What are the challenges in resolving potential experimental contradictions when studying MAV_0015 function?

Researchers studying MAV_0015 function may encounter contradictory experimental results due to:

• Expression system variations: Different expression systems (native versus recombinant) may yield proteins with different functional properties

• Tag interference: The N-terminal His tag used in recombinant expression may affect protein folding or function

• Membrane environment differences: The lipid composition of expression host membranes differs from native Mycobacterium avium membranes

• Strain-specific effects: Different M. avium strains may exhibit variations in MAV_0015 function

To address these contradictions, implement:

• Multiple complementary approaches for functional analysis

• Both in vitro and in vivo assays

• Controls using untagged protein where possible

• Validation in multiple M. avium strains

What is the optimal protocol for recombinant expression and purification of MAV_0015?

Based on available literature, the following protocol is recommended for recombinant expression and purification of MAV_0015:

• Expression system: E. coli with an N-terminal His tag fusion

• Expression construct: Full-length MAV_0015 (amino acids 1-93) in a suitable expression vector

• Induction conditions: Optimize IPTG concentration and temperature (typically 0.5-1 mM IPTG at 18-25°C)

• Lysis: Gentle membrane disruption to preserve protein structure

• Purification: Immobilized metal affinity chromatography (IMAC) using the N-terminal His tag

• Storage: As lyophilized powder or in Tris/PBS-based buffer with 6% trehalose, pH 8.0

• Reconstitution: In deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol for long-term storage at -20°C/-80°C

How can researchers effectively isolate and study MAV_0015 in its native membrane environment?

To study MAV_0015 in its native membrane environment:

• Membrane vesicle isolation: Generate giant plasma membrane vesicles (GPMVs) from cells expressing tagged MAV_0015

• Vesicle orientation: Ensure outside-out orientation of membrane proteins through selective enrichment using affinity-based methods

• Vesicle sonication: Convert GPMVs to small unilamellar vesicles (SUVs) for biophysical studies

• Affinity enrichment: Use tag-specific nanobodies to isolate vesicles containing MAV_0015

• Verification: Confirm protein presence and orientation using SDS-PAGE and Western blotting

• Functional assays: Perform assays directly on the membrane vesicles to maintain native lipid environment

Table 1: Comparison of Methods for Studying Membrane Proteins

What analytical techniques are most informative for characterizing MAV_0015 structure-function relationships?

To establish structure-function relationships for MAV_0015, employ the following analytical techniques:

• Circular dichroism (CD) spectroscopy: Determine secondary structure content and stability

• NMR spectroscopy: Analyze protein dynamics and ligand interactions

• Site-directed mutagenesis: Identify critical residues for function

• Cross-linking studies: Map protein-protein interaction interfaces

• Molecular dynamics simulations: Model protein behavior in membrane environments

• Functional assays: Measure cell division effects in complementation studies

• Immunoprecipitation: Identify interaction partners in vivo

For each technique, compare wild-type MAV_0015 with mutant variants targeting conserved residues to establish structure-function correlations

How should researchers analyze evolutionary conservation patterns of MAV_0015 across mycobacterial species?

A comprehensive evolutionary analysis of MAV_0015 should include:

• Multiple sequence alignment: Align MAV_0015 with orthologs from related species

• Phylogenetic tree construction: Use maximum likelihood or Bayesian methods

• Selection pressure analysis: Calculate dN/dS ratios to identify conserved functional regions

• Consensus sequence determination: Identify invariant residues across mycobacterial species

• Domain architecture analysis: Map conserved functional domains

Table 2: Key Orthologs of MAV_0015 (CrgA) Across Mycobacterial Species

What statistical approaches are most appropriate for analyzing MAV_0015 functional data in knockout versus complementation studies?

When analyzing functional data from MAV_0015 genetic manipulation studies:

• For growth rate comparisons:

  • Use repeated measures ANOVA with post-hoc tests

  • Apply Bonferroni correction for multiple comparisons

  • Consider mixed effects models for studies with random factors

• For cell morphology assessments:

  • Employ non-parametric tests (Mann-Whitney U) for non-normally distributed data

  • Use chi-square tests for categorical morphological classifications

  • Consider machine learning approaches for automated morphological analysis

• For gene expression studies:

  • Apply ANOVA or t-tests with appropriate multiple testing corrections

  • Use fold-change cutoffs in combination with statistical significance

  • Perform clustered analysis to identify co-regulated genes

• Data visualization recommendations:

  • Create box plots showing distribution of measurements

  • Use scatter plots with regression lines to show correlations

  • Generate heat maps for expression data across multiple conditions

How does MAV_0015 research contribute to understanding Mycobacterium avium complex infections and potential therapeutic approaches?

MAV_0015 research has significant implications for understanding and treating MAC infections:

• Infection mechanisms: As a cell division protein, MAV_0015 may be critical for bacterial replication during infection

• Therapeutic targeting: Essential bacterial proteins like MAV_0015 represent potential drug targets

• Diagnostic applications: MAV_0015-specific antibodies could be developed for diagnostic purposes

• Virulence connections: Understanding the relationship between cell division and virulence factors

• Drug resistance mechanisms: Potential connections between cell division proteins and antibiotic resistance

MAC infections are increasingly prevalent worldwide and often difficult to treat, requiring extended antibiotic regimens. They are particularly problematic in patients with underlying lung conditions or compromised immunity, with mortality rates of approximately 25% within 5 years. Research on essential proteins like MAV_0015 could lead to new therapeutic approaches for these challenging infections .

What future research directions should be prioritized to advance our understanding of MAV_0015 function?

Priority research directions for MAV_0015 should include:

• High-resolution structural studies: Determine the three-dimensional structure using cryo-EM or X-ray crystallography

• In vivo functional studies: Create conditional knockdowns to study essentiality in various growth conditions

• Protein-protein interaction network: Identify the complete interactome of MAV_0015

• Post-translational modifications: Characterize any modifications that regulate activity

• Drug discovery: Screen for small molecules that bind to and inhibit MAV_0015 function

• Structure-based mutagenesis: Create point mutations in key residues to map functional domains

• Comparative genomics: Study natural variations in clinical isolates and their functional consequences

• Systems biology approaches: Integrate transcriptomic and proteomic data to place MAV_0015 in broader cellular networks

What are common challenges in recombinant MAV_0015 expression and how can they be addressed?

When working with recombinant MAV_0015, researchers may encounter several challenges:

• Low expression yields:

  • Optimize codon usage for the expression host

  • Test multiple expression strains (BL21, C41/C43, Rosetta)

  • Reduce expression temperature (18°C overnight)

  • Try autoinduction media instead of IPTG induction

• Protein aggregation:

  • Include detergents (DDM, LDAO) during lysis

  • Test fusion partners (MBP, SUMO) to enhance solubility

  • Use specialized E. coli strains designed for membrane proteins

  • Apply on-column refolding protocols during purification

• Proteolytic degradation:

  • Include protease inhibitor cocktails in all buffers

  • Reduce purification time by optimizing protocols

  • Perform purification at 4°C

  • Consider adding stabilizing agents (glycerol, trehalose)

• Reconstitution issues:

  • Carefully control protein concentration during reconstitution (0.1-1.0 mg/mL)

  • Add glycerol (5-50% final concentration) to prevent aggregation

  • Avoid repeated freeze-thaw cycles

  • Store working aliquots at 4°C for up to one week

How can researchers validate the proper folding and functionality of purified MAV_0015?

To validate the proper folding and functionality of purified MAV_0015:

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Thermal shift assays to determine protein stability

  • Size exclusion chromatography to verify monodispersity

  • Dynamic light scattering to check for aggregation

• Functional verification:

  • Binding assays with known interaction partners

  • Complementation studies in knockout strains

  • Activity assays relevant to cell division function

• Structural integrity:

  • Limited proteolysis to verify compact folding

  • Native PAGE to assess oligomeric state

  • Intrinsic fluorescence to monitor tertiary structure

• Quality control metrics:

  • 90% purity as determined by SDS-PAGE

  • Single peak on size exclusion chromatography

  • Proper membrane insertion when reconstituted in liposomes

  • Consistent batch-to-batch functional activity

What are the key considerations for designing a comprehensive research program focused on MAV_0015?

A comprehensive research program on MAV_0015 should:

• Integrate multiple approaches: Combine structural, functional, and genetic methods

• Address clinical relevance: Connect fundamental research to MAC infection outcomes

• Employ comparative biology: Leverage knowledge from better-studied mycobacterial orthologs

• Develop specialized tools: Create antibodies, reporter strains, and expression systems

• Form collaborative networks: Engage experts in membrane protein biology, mycobacteriology, and structural biology

• Establish standardized protocols: Develop reproducible methods for expression and functional characterization

• Consider translational aspects: Identify paths to therapeutic or diagnostic applications