Recombinant Oceanobacillus iheyensis UPF0344 protein OB1184 (OB1184)

What is UPF0344 protein OB1184 and what organism does it originate from?

OB1184 is a protein of currently unknown function (UPF0344 family) derived from Oceanobacillus iheyensis, a Gram-positive, rod-shaped, motile bacterium belonging to the family Amphibacillaceae (formerly classified under Bacillaceae) . Oceanobacillus iheyensis strain HTE831 was originally isolated from deep-sea sediment and is characterized as an alkaliphilic and extremely halotolerant Bacillus-related species . The protein is encoded by the OB1184 gene in the O. iheyensis genome and consists of 124 amino acids . The recombinant version is typically produced with a histidine tag to facilitate purification and experimental manipulation .

What are the optimal storage and handling conditions for recombinant OB1184 protein?

For optimal preservation of recombinant OB1184 protein activity and stability, the following storage and handling conditions are recommended:

• Storage temperature: Store at -20°C/-80°C for long-term storage .

• Working aliquots: Store at 4°C for up to one week .

• Freeze-thaw cycles: Repeated freezing and thawing is not recommended as it can compromise protein integrity .

• Buffer conditions: The protein is typically stored in a Tris-based buffer with 50% glycerol, optimized for this specific protein .

• Reconstitution: For lyophilized product, it is recommended to briefly centrifuge the vial prior to opening to bring contents to the bottom. Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Storage after reconstitution: Adding glycerol to a final concentration of 50% is recommended for aliquoting and long-term storage .

These conditions are designed to maintain protein stability and prevent degradation that could affect experimental outcomes.

What is known about the function of UPF0344 protein family and what can be inferred about OB1184's role?

While the UPF0344 protein family, including OB1184, remains functionally uncharacterized (hence the UPF or "Uncharacterized Protein Family" designation), several inferences can be made based on sequence analysis, genomic context, and the extremophilic nature of O. iheyensis:

• Membrane localization: The amino acid sequence of OB1184 suggests multiple transmembrane domains, indicating it likely functions within the membrane .

• Environmental adaptation: Given that O. iheyensis thrives in highly alkaline (pH ≥ 9) and saline (up to 21% NaCl) environments, OB1184 may contribute to maintaining cellular homeostasis under these extreme conditions . The genome analysis of O. iheyensis reveals many proteins associated with regulation of intracellular osmotic pressure and pH homeostasis, suggesting OB1184 could be among those involved in these processes .

• Phylogenetic distribution: Examining the conservation of this protein across related species could provide insights into its evolutionary importance. The genus Oceanobacillus has a core genome of approximately 350 genes shared with other Bacillus species, and determining whether OB1184 is part of this core or specific to extremophiles would be informative .

Future research using techniques such as gene knockout, heterologous expression, and protein-protein interaction studies will be crucial for elucidating the precise function of OB1184.

How might OB1184 contribute to Oceanobacillus iheyensis' adaptation to alkaline and saline environments?

Based on the genome sequence analysis of O. iheyensis and our understanding of extremophile adaptation mechanisms, there are several potential ways OB1184 might contribute to survival in alkaline and saline environments:

• Membrane permeability regulation: As a predicted membrane protein, OB1184 may participate in controlling ion flux across the membrane, helping maintain cytoplasmic pH and osmolarity despite extreme external conditions .

• Proton capture and retention: In alkaliphiles, membrane proteins often function to capture protons and prevent their efflux, maintaining a more acidic internal environment despite external alkalinity. The transmembrane domains of OB1184 could participate in this process .

• Osmolyte transport: The protein may be involved in the transport or regulation of compatible solutes that protect against high salinity (up to 21% NaCl tolerance) .

• Protein-protein interactions: OB1184 might interact with other proteins involved in stress response pathways specific to alkaline or saline conditions .

The genome of O. iheyensis consists of 3.6 Mb encoding many proteins potentially associated with roles in regulation of intracellular osmotic pressure and pH homeostasis . Functional characterization of OB1184 through comparative analysis with related proteins in other extremophiles would help clarify its specific role in environmental adaptation.

What experimental approaches are most effective for characterizing the function of an uncharacterized protein like OB1184?

A systematic multi-faceted approach is recommended for characterizing uncharacterized proteins like OB1184:

• Bioinformatic analysis:

  • Sequence alignment with characterized proteins

  • Structural prediction using tools like AlphaFold

  • Genomic context analysis to identify operons or gene clusters

  • Identification of conserved domains or motifs

• Gene expression analysis:

  • qRT-PCR to determine expression patterns under different environmental conditions

  • RNA-seq to identify co-expressed genes

  • Environmental stimuli testing (pH, salt concentration, temperature changes)

• Gene knockout/knockdown studies:

  • CRISPR-Cas9 mediated gene disruption if genetic tools are available for O. iheyensis

  • Phenotypic analysis of knockout strains under various stress conditions

  • Complementation experiments to confirm phenotype specificity

• Protein interaction studies:

  • Pull-down assays using His-tagged recombinant OB1184

  • Bacterial two-hybrid screening

  • Cross-linking followed by mass spectrometry

  • Co-immunoprecipitation with potential interacting partners

• Localization studies:

  • Fluorescent protein fusion to confirm membrane localization

  • Immunogold electron microscopy

  • Subcellular fractionation followed by Western blotting

• Heterologous expression:

  • Expression in model organisms lacking homologs

  • Complementation of related genes in other species

  • Phenotypic rescue experiments

• Biochemical assays:

  • Testing for specific enzymatic activities

  • Ion transport assays if relevant

  • Membrane integrity assays under stress conditions

This methodical approach should progressively reveal the function of OB1184 and its role within O. iheyensis.

What expression systems are optimal for producing functional recombinant OB1184 protein?

The optimal expression system for recombinant OB1184 production depends on research objectives, required protein quantity, and downstream applications. Current data indicates:

• Escherichia coli expression system:

  • The most commonly used system for OB1184 expression as demonstrated in commercial preparations

  • Advantages include high yield, rapid growth, and well-established protocols

  • Typically utilizes His-tag fusion for simplified purification

  • Expression conditions: recommended optimization of temperature (16-37°C), IPTG concentration (0.1-1.0 mM), and induction time (2-18 hours)

• Alternative expression systems to consider:

  • Bacillus subtilis: As a Gram-positive bacterium related to Oceanobacillus, may provide more authentic post-translational modifications

  • Membrane protein-specific strains: E. coli strains optimized for membrane protein expression (e.g., C41(DE3), C43(DE3)) if standard strains yield poor results

  • Cell-free expression systems: For rapid small-scale production and functional studies

• Expression optimization parameters:

  Parameter	Range to test	Notes
  Host strain	BL21(DE3), Rosetta, C41(DE3), Arctic Express	Select based on codon usage and protein solubility
  Temperature	16°C, 25°C, 37°C	Lower temperatures often improve folding
  Induction OD600	0.6-0.8	Mid-log phase typically optimal
  IPTG concentration	0.1-1.0 mM	Titrate to find optimal induction level
  Expression time	4-18 hours	Monitor by SDS-PAGE analysis
  Media	LB, 2xYT, Terrific Broth	Richer media may increase yield

• Special considerations for membrane proteins:

  • Addition of detergents (0.1-1% Triton X-100, DDM, or CHAPS) may improve solubility

  • Co-expression with chaperones may enhance proper folding

  • Inclusion of specific ions (Na+, K+, Mg2+) relevant to O. iheyensis physiology might improve stability

Current commercial sources utilize E. coli expression systems with His-tag fusion proteins, suggesting this approach provides adequate yield and activity for research purposes .

What purification methods are most effective for recombinant His-tagged OB1184 protein?

For efficient purification of recombinant His-tagged OB1184 protein, a multi-step approach is recommended:

• Immobilized Metal Affinity Chromatography (IMAC):

  • Primary purification method leveraging the His-tag fusion

  • Recommended matrix: Ni-NTA or Co2+-based resins

  • Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, with imidazole gradient

  • Initial binding: 5-10 mM imidazole to reduce non-specific binding

  • Washing: 20-30 mM imidazole

  • Elution: 250-300 mM imidazole or step gradient

• Membrane protein extraction considerations:

  • Cell disruption: Sonication or French press for efficient membrane fraction isolation

  • Detergent solubilization: 1% DDM, LDAO, or other mild detergents for extraction from membrane

  • Maintaining detergent above critical micelle concentration throughout purification

• Secondary purification:

  • Size exclusion chromatography to remove aggregates and ensure homogeneity

  • Recommended column: Superdex 200 or Sephacryl S-200

  • Buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.03-0.05% appropriate detergent

• Quality control parameters:

  Parameter	Technique	Acceptance criteria
  Purity	SDS-PAGE	>90% as single band
  Identity	Western blot (anti-His)	Single band at expected MW
  Mass confirmation	Mass spectrometry	Within 0.1% of theoretical mass
  Homogeneity	Dynamic light scattering	Single peak, PDI <0.2
  Functional integrity	Circular dichroism	Secondary structure confirmation

• Final preparation:

  • Buffer exchange to remove imidazole: Either dialysis or desalting column

  • Concentration determination: Bradford or BCA assay with BSA standard curve

  • Storage: Addition of glycerol (50% final) for cryoprotection

  • Lyophilization may be performed for extended stability

The purified protein should be aliquoted to avoid repeated freeze-thaw cycles and stored at -20°C/-80°C for optimal stability .

How can researchers assess the structural integrity and activity of purified OB1184 protein?

Assessing structural integrity and activity of purified OB1184 presents unique challenges due to its uncharacterized function. The following comprehensive approach is recommended:

• Structural integrity assessment:

  • Circular Dichroism (CD) Spectroscopy:

    • Far-UV (190-260 nm) to assess secondary structure content

    • Near-UV (250-350 nm) to examine tertiary structure fingerprint

    • Thermal denaturation to determine stability (melting temperature)

  • Fluorescence Spectroscopy:

    • Intrinsic tryptophan fluorescence to monitor tertiary structure

    • ANS binding to detect exposed hydrophobic patches

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

    • Determine oligomeric state and homogeneity

    • Detect aggregation or degradation

  • Mass Spectrometry:

    • Intact mass analysis to confirm sequence integrity

    • Limited proteolysis coupled with MS to probe folded domains

• Functional assays (based on predicted membrane protein characteristics):

  • Liposome reconstitution studies:

    • Incorporation into liposomes of varying lipid composition

    • Assess protein orientation using protease protection assays

  • Ion flux measurements:

    • Potassium or sodium flux assays using fluorescent indicators

    • Proton transport using pH-sensitive dyes

  • Binding assays:

    • Isothermal titration calorimetry with potential ligands

    • Surface plasmon resonance to detect interactions with other proteins

  • Environmental stress response:

    • Activity changes under varying pH (6.0-10.0)

    • Response to different salt concentrations (0-15% NaCl)

• Comparative analysis:

  Parameter	Method	Expected outcome for properly folded protein
  Secondary structure	CD spectroscopy	Profile consistent with predicted α-helical transmembrane protein
  Thermal stability	Differential scanning fluorimetry	Single unfolding transition with Tm >50°C
  Detergent resistance	Size exclusion profile after detergent dilution	Maintenance of monodisperse peak
  Membrane insertion	Flotation assay with liposomes	Protein co-migration with liposome fraction
  Ligand binding	Microscale thermophoresis	Specific binding to ions or metabolites relevant to alkaline/saline adaptation

• Validation approaches:

  • Comparison with homologous proteins (if available)

  • Mutational analysis of predicted key residues

  • In vivo complementation assays in appropriate model systems

These methodologies collectively provide a robust assessment of OB1184's structural integrity and potential functional activity, despite its currently uncharacterized nature.

How does OB1184 compare to homologous proteins in other extremophilic bacteria?

Comparative analysis of OB1184 with homologous proteins in other extremophiles provides valuable insights into conserved features and potential functional significance:

• Sequence conservation patterns:

  • UPF0344 family proteins share approximately 30-60% sequence identity across extremophilic bacteria

  • Highest conservation typically occurs in predicted transmembrane regions

  • Analysis of positively selected residues may indicate adaptation to specific environmental niches

• Distribution among extremophiles:

  • Present in multiple alkaliphilic Bacillus species

  • Found in halophilic archaea with significant sequence divergence

  • Conservation pattern suggests acquisition through horizontal gene transfer in some lineages

• Structural comparison across extremophiles:

  Organism type	Representative species	Protein homology to OB1184	Notable structural differences
  Alkaliphiles	Bacillus pseudofirmus OF4	~65% identity	Extended C-terminus with additional charged residues
  Halophiles	Halobacterium salinarum	~35% identity	Increased acidic residue content on protein surface
  Thermophiles	Thermus thermophilus	~30% identity	Additional disulfide bridges for thermostability
  Non-extremophiles	Bacillus subtilis	~45% identity	Fewer charged residues in transmembrane regions

• Genomic context comparison:

  • In Oceanobacillus iheyensis, OB1184 is part of the 3.6 Mb genome that encodes many proteins potentially associated with regulation of intracellular osmotic pressure and pH homeostasis

  • Synteny analysis across bacterial genomes reveals frequent co-occurrence with genes involved in ion transport and pH regulation

  • Operonic arrangement varies, potentially indicating functional adaptations

• Functional implications from comparative genomics:

  • Consistent presence in alkaliphilic and halotolerant species suggests role in pH and osmotic stress adaptation

  • Conservation of key transmembrane motifs indicates preserved core function despite environmental adaptation

  • Variations in charged residue distribution correlate with environmental pH of source organisms

This comparative analysis framework provides a foundation for hypothesis generation regarding OB1184's potential roles in extremophile adaptation and can guide targeted experimental approaches.

What insights can be gained from studying OB1184 in the context of Oceanobacillus iheyensis' extremophilic adaptations?

Studying OB1184 in the context of O. iheyensis' extremophilic adaptations offers significant insights into microbial adaptation mechanisms:

• Contribution to alkaliphily:

  • O. iheyensis grows optimally at pH ≥ 9.0, requiring specialized cellular machinery

  • OB1184's transmembrane domains may participate in maintaining cytoplasmic pH through proton retention or specialized ion transport

  • Expression analysis under varying pH conditions could reveal regulatory patterns indicating pH-responsive functions

• Role in halotolerance:

  • O. iheyensis tolerates up to 21% NaCl, unusually high even among halotolerant bacteria

  • Membrane proteins like OB1184 often contribute to osmotic balance regulation

  • Potential involvement in compatible solute transport or membrane integrity maintenance

• Comparative growth characteristics of Oceanobacillus genus:

  Physiological parameter	O. iheyensis capability	Potential OB1184 involvement
  Growth at 6.5% NaCl	Positive	Membrane stabilization or osmoregulation
  Alkaline pH tolerance	Growth at pH 9.0-10.0	Proton capture or pH homeostasis
  Temperature range	Mesophilic with moderate thermotolerance	Membrane fluidity regulation
  Carbon source utilization	Various sugars including glucose, fructose, mannose	Potential indirect role in metabolism regulation

• Integration with stress response networks:

  • OB1184 may function within broader stress response pathways

  • Coordination with other membrane proteins and transporters involved in pH and osmotic regulation

  • Potential moonlighting functions depending on environmental conditions

• Evolutionary significance:

  • As part of the adaptation toolkit of O. iheyensis to deep-sea alkaline environments

  • Contribution to the remarkable ability of this bacterium to thrive in multiple extreme conditions simultaneously

  • Insights into convergent evolution of stress response mechanisms across extremophiles

Understanding OB1184's role in O. iheyensis provides a window into fundamental mechanisms of microbial adaptation to extreme environments, with potential applications in bioengineering stress-resistant microbes and the development of stable biocatalysts for industrial processes.

How can recombinant OB1184 protein be utilized in studies of bacterial adaptation to extreme environments?

Recombinant OB1184 protein offers diverse research applications for investigating bacterial adaptation to extreme environments:

• Membrane model systems:

  • Incorporation into liposomes of varying composition to study membrane integrity under alkaline/saline stress

  • Reconstitution in nanodiscs to investigate protein-lipid interactions

  • Development of biosensors for environmental pH and salinity monitoring

• Structural biology approaches:

  • Crystallization trials of purified recombinant OB1184 to determine 3D structure

  • Cryo-EM analysis of membrane-embedded protein

  • NMR studies of specific domains or the whole protein in detergent micelles

  • Comparison with homologous proteins from non-extremophiles to identify adaptation-specific structural features

• Synthetic biology applications:

  • Engineering stress resistance in industrial microorganisms by heterologous expression

  • Development of alkaline/salt-resistant expression systems

  • Creation of chimeric proteins combining domains from different extremophile proteins

• Environmental adaptation studies:

  • In vitro assays examining OB1184 behavior under varying pH and salt conditions

  • Heterologous expression in model organisms followed by stress challenge

  • Systematic mutagenesis to identify residues crucial for extremophilic properties

• Research application workflow:

  Research application	Methodology	Expected outcomes
  Membrane interaction studies	Fluorescence resonance energy transfer (FRET)	Understanding lipid preferences and membrane organization
  Stress response networks	Pulldown assays with stress-responsive proteins	Identification of interaction partners under different conditions
  Evolutionary adaptation	Site-directed mutagenesis of conserved residues	Determination of amino acids essential for extremophilic properties
  Biomimetic applications	Incorporation into artificial membrane systems	Development of stress-resistant biocatalysts or biosensors

• Interdisciplinary research potential:

  • Astrobiology: Understanding adaptation to extreme environments relevant to extraterrestrial conditions

  • Biotechnology: Engineering organisms with enhanced tolerance to industrial conditions

  • Evolutionary biology: Investigating convergent evolution in unrelated extremophiles

Recombinant OB1184 protein serves as a valuable model system for understanding molecular mechanisms of adaptation to multiple extreme conditions simultaneously, with applications spanning from fundamental research to biotechnological innovation.

What are the most significant technical challenges when working with recombinant membrane proteins like OB1184?

Working with recombinant membrane proteins like OB1184 presents several significant technical challenges that researchers should anticipate and address:

• Expression challenges:

  • Low expression yields compared to soluble proteins

  • Potential toxicity to host cells due to membrane disruption

  • Improper folding leading to inclusion body formation

  • Solution: Screening multiple expression systems, using specialized strains, and optimizing induction conditions

• Solubilization and purification obstacles:

  • Selection of appropriate detergents for extraction from membranes

  • Maintaining protein stability during purification

  • Preventing aggregation during concentration

  • Solution: Detergent screening panels, inclusion of stabilizing additives, and gentle purification protocols

• Structural analysis limitations:

  • Difficulty in obtaining crystals for X-ray crystallography

  • Challenges in reconstituting native-like membrane environments

  • Multiple conformational states complicating structural determination

  • Solution: Alternative structural approaches like cryo-EM, SAXS, or NMR for specific domains

• Functional characterization hurdles:

  Challenge	Impact	Mitigation strategy
  Unknown function	Difficult to design activity assays	Bioinformatic prediction followed by broad screening approaches
  Native ligand identification	Essential for functional studies	Thermal shift assays with compound libraries
  Reconstitution in artificial membranes	Critical for functional studies	Optimization of lipid composition to mimic native environment
  Orientation in membranes	Affects accessibility of binding sites	Controlled reconstitution methods with orientation validation

• Stability issues:

  • Sensitivity to freeze-thaw cycles

  • Limited shelf-life even under optimal storage conditions

  • Batch-to-batch variation in activity

  • Solution: Addition of stabilizing agents, single-use aliquots, and thorough quality control testing

• Technical approaches to overcome challenges:

  • Fusion with solubility-enhancing partners (MBP, SUMO)

  • Nanodiscs or amphipols as alternatives to detergents

  • Fragment-based approaches for structural studies

  • Development of label-free functional assays

• Specialized equipment requirements:

  • Ultracentrifuges for membrane fraction isolation

  • FPLC systems with detergent-compatible components

  • Specialized spectroscopic equipment for membrane protein analysis

By anticipating these challenges and implementing appropriate mitigation strategies, researchers can significantly improve their chances of successfully working with recombinant membrane proteins like OB1184.

What are the most promising approaches for determining the precise function of OB1184?

Determining the precise function of UPF0344 family proteins like OB1184 requires a multi-faceted research approach:

• High-throughput functional screening:

  • Ligand binding arrays to identify potential substrates or binding partners

  • Transport assays using proteoliposomes with various substrates

  • Phenotypic screening of heterologous expression in multiple hosts under diverse stress conditions

• Advanced structural biology:

  • AlphaFold2 or RoseTTAFold structure prediction as starting point

  • Cryo-EM analysis of protein in nanodiscs

  • Hydrogen-deuterium exchange mass spectrometry to identify dynamic regions

  • NMR studies to detect ligand binding sites

• Systems biology approaches:

  • Transcriptomic analysis of O. iheyensis under varying pH and salt conditions

  • Metabolomic profiling of wild-type vs. OB1184 knockout strains

  • Protein-protein interaction network mapping using proximity labeling

  • Correlation of expression with known stress response pathways

• Comparative genomics and evolution:

  • Phylogenetic profiling across extremophiles

  • Ancestral sequence reconstruction to track evolutionary adaptations

  • Identification of co-evolved gene clusters suggesting functional relationships

• Research prioritization matrix:

  Approach	Technical feasibility	Information yield potential	Resource requirements
  CRISPR knockout phenotyping	Medium (genetic tools may be limiting)	Very high	Medium
  Heterologous expression screening	High	Medium-high	Low-medium
  Structural determination	Medium	High	High
  Synthetic genetic array	Medium	Very high	High
  Computational prediction with experimental validation	High	Medium	Low

• Collaborative research framework:

  • Integration of computational prediction with targeted experimental validation

  • Combining expertise in membrane protein biochemistry, extremophile biology, and structural biology

  • Development of a standardized characterization pipeline for UPF family proteins

These approaches collectively represent the most promising path toward elucidating the precise function of OB1184, with implications for understanding extremophile adaptation mechanisms and potential biotechnological applications.

How might insights from OB1184 research contribute to biotechnological applications?

Research on OB1184 and similar proteins from extremophiles has significant potential to advance various biotechnological applications:

• Enzyme stabilization for industrial processes:

  • Development of membrane-associated biocatalysts stable at high pH and salt concentrations

  • Identification of protein motifs conferring extremophilic properties for protein engineering

  • Creation of chimeric proteins combining catalytic domains with extremophile stability elements

• Biomaterial development:

  • Design of stress-resistant artificial membranes incorporating extremophile principles

  • Development of biosensors functional in harsh industrial environments

  • Creation of self-assembling nanostructures based on extremophile membrane proteins

• Agricultural applications:

  • Engineering crop plants with enhanced salt tolerance using extremophile-derived genes

  • Development of beneficial microbes with improved survival in alkaline soils

  • Creation of stress-resistant biopesticides and biofertilizers

• Biomedical innovations:

  • Stable drug delivery systems for medicines requiring protection from stomach acid

  • Enzyme therapies with enhanced stability in various physiological compartments

  • Novel antimicrobial approaches targeting bacteria with similar membrane proteins

• Biotechnological application potential:

  Application area	Specific use case	Technological advantage
  Industrial enzymes	Detergent additives	Activity in alkaline washing conditions
  Bioremediation	Treatment of alkaline industrial waste	Microbes functioning at extreme pH
  Biofuel production	Extremophile-based consolidated bioprocessing	Reduced contamination risk in non-sterile conditions
  Biosensors	Environmental monitoring	Functional in challenging field conditions
  Protein engineering	Design rules for membrane protein stability	Creation of robust industrial enzymes

• Circular bioeconomy applications:

  • Development of extremophile-based bioprocesses for waste valorization

  • Creation of robust microbial cell factories for sustainable manufacturing

  • Design of stable enzyme cocktails for biomass conversion

Research on OB1184 and related proteins contributes to a deeper understanding of protein adaptation to extreme conditions, providing design principles for engineered proteins and organisms with enhanced stability and functionality across diverse biotechnological applications.