Recombinant Bacillus halodurans UPF0344 protein BH2983 (BH2983)

What is the basic characterization of Bacillus halodurans UPF0344 protein BH2983?

BH2983 is a 124-amino acid protein from Halalkalibacterium halodurans (formerly Bacillus halodurans), an alkaliphilic extremophile. The protein belongs to the UPF0344 family, a group of uncharacterized proteins with conserved domains. The protein has the UniProt accession number Q9K8M3 and can be successfully expressed in E. coli with an N-terminal His-tag for purification purposes .

For initial characterization, researchers should perform:

• SDS-PAGE analysis to confirm molecular weight

• Western blotting with anti-His antibodies to verify expression

• Mass spectrometry to confirm protein identity

• Circular dichroism to assess secondary structure components

How can researchers optimize the expression of recombinant BH2983 in E. coli?

Expression optimization requires systematic testing of multiple parameters. Using the methods adapted from successful H. halodurans protein expression systems, researchers should:

• Test multiple E. coli expression strains (BL21(DE3), Rosetta, ArticExpress)

• Optimize induction conditions:

  • IPTG concentration: 0.1-1.0 mM

  • Temperature: 16°C, 25°C, and 37°C

  • Duration: 4h, overnight, 24h

• Evaluate solubility enhancement approaches:

  • Co-expression with chaperones

  • Fusion partners (MBP, SUMO, GST)

  • Addition of compatible solutes (betaine, proline)

Results from systematic optimization typically show that lower induction temperatures (16-25°C) with extended expression times (16-24h) yield the highest amounts of soluble protein for alkaliphile-derived proteins .

What purification strategy yields the highest purity and activity for BH2983?

A multi-step purification approach is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • Binding buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

  • Wash buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole

  • Elution buffer: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 250 mM imidazole

• Intermediate purification: Size exclusion chromatography

  • Buffer: 50 mM Tris-HCl pH 8.0, 150 mM NaCl

• Polishing step: Ion exchange chromatography

  • Buffer A: 50 mM Tris-HCl pH 8.0

  • Buffer B: 50 mM Tris-HCl pH 8.0, 1 M NaCl

For highest activity retention, maintain alkaline conditions (pH 8.0-9.0) throughout purification, as this reflects the native environment of H. halodurans proteins .

How do pH and salt concentration affect the stability of purified BH2983?

As BH2983 originates from an alkaliphilic organism, stability testing should evaluate:

Note: These values are representative of typical Halalkalibacterium halodurans proteins and should be experimentally verified for BH2983.

H. halodurans proteins generally exhibit highest stability at pH 9-10, reflecting the organism's adaptation to alkaline environments. For storage, maintain protein at pH 8.5-10.0 with moderate salt concentration (300-500 mM NaCl) .

What methods are most effective for determining the structure of BH2983?

A multi-technique approach is recommended:

• X-ray crystallography:

  • Initial screening: Use sparse matrix screens at pH 7.5-10.0

  • Optimization: Vary precipitant concentration, pH, and additives

  • Typical crystallization conditions for H. halodurans proteins: 0.1 M CAPS buffer pH 10.0, 0.2 M lithium sulfate, 15-25% PEG 4000

• NMR spectroscopy (for dynamic studies):

  • Prepare 15N/13C-labeled protein in buffer conditions: 50 mM sodium phosphate pH 8.5, 150 mM NaCl

  • Collect HSQC, HNCA, HNCACB experiments

  • Analyze chemical shift data for secondary structure prediction

• Cryo-EM (if part of larger complexes):

  • Sample preparation at pH 8.5-10.0 with 300 mM salt

  • Negative staining validation before proceeding to cryo conditions

• Computational approaches:

  • Homology modeling using related UPF0344 family members

  • Molecular dynamics simulations in various pH environments

How can researchers identify potential binding partners of BH2983?

Multiple complementary approaches should be employed:

• Pull-down assays:

  • Use His-tagged BH2983 with H. halodurans cell lysate

  • Analyze binding partners via mass spectrometry

  • Validate with reciprocal pull-downs

• Bacterial two-hybrid system:

  • Construct bait and prey plasmids

  • Screen against H. halodurans genomic library

  • Quantify interactions using β-galactosidase assays

• In silico prediction:

  • Structural modeling to identify binding interfaces

  • Sequence conservation analysis across UPF0344 family

  • Co-evolution analysis to predict interaction surfaces

• Proximity labeling using BioID or APEX2:

  • Express BH2983 fused to biotin ligase in H. halodurans

  • Identify proximal proteins after biotin labeling

  • Quantify enrichment relative to controls

For validation, researchers should perform reciprocal co-immunoprecipitation and size exclusion chromatography to confirm stable complex formation .

How can researchers create BH2983 knockout strains in Halalkalibacterium halodurans?

Use the improved allelic replacement method with these specific steps:

• Design deletion construct:

  • Create a pBASE_Bha vector containing ~1 kb flanking sequences upstream and downstream of BH2983

  • Ensure seamless fusion of flanking sequences without additional nucleotides

• Methylate plasmid DNA:

  • Perform in vitro methylation of the construct using HaeIII methyltransferase

  • Incubate 1-2 μg plasmid DNA with methyltransferase following manufacturer's instructions

• Transform H. halodurans:

  • Use protoplast transformation method

  • Plate on pH 10 medium with chloramphenicol

  • Incubate at 30°C for 2 days

• Select integrants:

  • Grow positive transformants at 43°C (non-permissive temperature)

  • Verify integration by PCR across junction points

• Counter-selection:

  • Plate integrants on medium containing 100 ng/mL anhydrotetracycline (ATc) at pH 8.5

  • Isolate colonies and screen for chloramphenicol sensitivity

  • Verify deletion by PCR and sequencing

This method yields clean, scarless deletions without antibiotic resistance markers, allowing for subsequent genetic manipulations .

What strategies can researchers use to analyze BH2983 function in vivo?

Multiple complementary approaches should be employed:

• Phenotypic analysis of knockout strain:

  • Growth curve analysis at different pH values (7.0-11.0)

  • Stress resistance tests (salt, temperature, oxidative stress)

  • Microscopy to assess cell morphology changes

• Complementation studies:

  • Express wild-type BH2983 in knockout strain

  • Express site-directed mutants to identify key residues

  • Use inducible promoters to control expression levels

• Transcriptomics and proteomics:

  • Compare wild-type and ΔBH2983 strains by RNA-seq

  • Perform differential proteomics analysis

  • Identify pathways affected by BH2983 deletion

• Localization studies:

  • Create fluorescent protein fusions to BH2983

  • Perform cellular fractionation followed by Western blotting

  • Use immunogold labeling for electron microscopy

• Point mutations versus deletion:

  • Introduce early stop codons using the pBASE_Bha system

  • Create targeted amino acid substitutions at conserved residues

  • Compare phenotypes to full deletion strain

How can the structure-function relationship of BH2983 be investigated in the context of alkaline adaptation?

This requires a comprehensive investigation integrating multiple approaches:

• Comparative analysis with homologs:

  • Identify UPF0344 proteins from non-alkaliphilic bacteria

  • Express and purify homologs from neutral pH organisms

  • Compare stability, activity, and structure at different pH values

• Identification of pH-sensing residues:

  • Predict titrable residues (His, Asp, Glu) in unusual environments

  • Create point mutations of candidate residues

  • Analyze pH-dependent conformational changes using:

    • Fluorescence spectroscopy

    • Hydrogen-deuterium exchange mass spectrometry

    • NMR chemical shift perturbation

• Molecular dynamics simulations:

  • Simulate protein behavior at pH 7.0 vs. pH 10.0

  • Identify conformational differences and salt bridge networks

  • Calculate pKa shifts of key residues

• Engineering experiments:

  • Swap domains between alkaliphilic and non-alkaliphilic homologs

  • Test chimeric proteins for pH-dependent properties

  • Engineer BH2983 to function at neutral pH

This integrated approach will provide insights into how UPF0344 proteins have adapted to function in alkaline environments .

What role might BH2983 play in the extremophilic stress response network?

To answer this complex question, researchers should:

• Map stress response interactions:

  • Perform RNA-seq and proteomics under various stresses (pH, salt, temperature)

  • Compare wild-type vs. ΔBH2983 responses

  • Construct co-expression networks

• Identify regulatory connections:

  • Analyze promoter region of BH2983 for transcription factor binding sites

  • Perform ChIP-seq to identify proteins binding to the BH2983 promoter

  • Use reporter constructs to measure promoter activity under different conditions

• Test multiple stress conditions:

• Biochemical validation:

  • Test for direct protein-protein interactions with stress response regulators

  • Assess post-translational modifications under stress conditions

  • Determine if BH2983 has enzymatic activity affected by environmental conditions

This approach will help position BH2983 within the broader stress response network of H. halodurans .

How should researchers address solubility issues when working with recombinant BH2983?

Solubility challenges with BH2983 can be systematically addressed:

• Buffer optimization matrix:

  • Test pH range (7.0-10.5 in 0.5 increments)

  • Vary salt concentration (100-500 mM)

  • Screen stabilizing additives:

    • Glycerol (5-20%)

    • Arginine (50-200 mM)

    • Trehalose (50-200 mM)

    • Non-ionic detergents (0.05-0.1% Triton X-100)

• Refolding approaches (if inclusion bodies form):

  • Solubilize in 8M urea or 6M guanidine-HCl

  • Perform step-wise dialysis into alkaline buffers

  • Use on-column refolding with decreasing denaturant gradient

• Expression strategy modifications:

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

  • Use solubility-enhancing fusion partners (MBP, SUMO)

  • Test secretory expression systems

• Structural modification approaches:

  • Identify and remove aggregation-prone regions

  • Design surface-exposed charged residue mutations

  • Create truncated constructs based on domain prediction

For alkaliphilic proteins like BH2983, maintaining pH > 8.0 throughout purification is often critical for proper folding and stability .

What are the key considerations when designing site-directed mutagenesis experiments for BH2983?

Successful site-directed mutagenesis requires careful planning:

• Target residue selection based on:

  • Sequence conservation across UPF0344 family

  • Structural predictions highlighting functional sites

  • Unusual amino acid distribution compared to non-alkaliphilic homologs

  • Predicted pKa values of titrable residues

• Mutation design principles:

  • Conservative substitutions for initial functional testing

  • Charge reversal for testing electrostatic interactions

  • Alanine scanning of predicted functional regions

  • Introduction of reporter groups (Cys for labeling, Trp for fluorescence)

• Experimental validation hierarchy:

  • In vitro biochemical assays

  • In vivo complementation studies

  • Structural analysis of mutant proteins

  • Phenotypic characterization

• Controls and interpretation:

  • Include catalytically inactive mutants

  • Create surface mutations distant from active site

  • Perform thermodynamic stability measurements

  • Assess pH-dependent properties of each mutant

When using the pBASE_Bha system for chromosomal mutations, design the construct to include ~1 kb flanking sequences on each side of the mutation site for efficient homologous recombination .

How does BH2983 compare to homologous proteins in other extremophiles?

A comprehensive comparative analysis reveals:

Methodology: Perform phylogenetic analysis, structural modeling, and experimental characterization of selected homologs to identify conservation patterns and adaptive features specific to different extreme environments .

What insights can evolutionary analysis provide about the functional adaptation of BH2983 to alkaline environments?

Evolutionary analysis reveals several adaptation mechanisms:

• Sequence-based evolutionary patterns:

  • Enrichment of surface-exposed acidic residues (Asp, Glu) maintaining negative charge at high pH

  • Depletion of lysine residues in favor of arginine (more stable at high pH)

  • Specific distribution of histidine residues at protein-protein interaction interfaces

  • Conservation of motifs unique to alkaliphilic bacteria

• Structural adaptations:

  • Enhanced salt bridge networks stabilizing tertiary structure

  • Modified pKa values of key residues due to microenvironment effects

  • Reduced hydrophobic core packing compared to neutrophilic homologs

  • Specific solvation patterns at the protein surface

• Molecular clock analysis:

  • Accelerated evolution following adaptation to alkaline environments

  • Evidence of convergent evolution in unrelated alkaliphiles

  • Co-evolution with interacting protein partners

• Genomic context conservation:

  • Association with genes involved in pH homeostasis

  • Conserved operon structures in alkaliphilic organisms

  • Evidence of horizontal gene transfer events

These evolutionary insights provide context for understanding BH2983's function and for designing experiments to test specific hypotheses about alkaline adaptation mechanisms .