Recombinant Bacillus licheniformis UPF0637 protein BLi01683/BL05149 (BLi01683, BL05149)

What is known about the molecular characteristics of UPF0637 protein BLi01683/BL05149?

UPF0637 protein BLi01683/BL05149 is classified as part of an uncharacterized protein family (UPF) in Bacillus licheniformis. While its specific function remains to be fully elucidated, structural analysis indicates it belongs to a conserved protein family present in various bacterial species. The protein is believed to contribute to B. licheniformis' remarkable adaptability and survival in diverse environments .

Methodology for initial characterization typically involves:

• Sequence alignment with homologous proteins using BLAST or similar tools

• Secondary structure prediction using algorithms such as PSIPRED

• Domain analysis through tools like SMART or Pfam

• Analysis of conserved motifs using MEME or related software

The protein likely adopts a specific structural conformation that enables its biological function, which may be related to the antimicrobial properties exhibited by B. licheniformis, as this bacterium is known to produce various bioactive compounds including bacteriocins and antimicrobial peptides .

What expression systems are most effective for producing recombinant UPF0637 protein?

The optimal expression system for UPF0637 protein depends on your experimental goals, but several approaches have proven effective:

Table 1: Comparison of Expression Systems for Recombinant B. licheniformis Proteins

For homologous expression in B. licheniformis, the pHY300PLK expression system has been effectively used for other recombinant proteins with excellent results . Recent advances using multiple ribosomal binding sites (RBSs) within a single mRNA leader sequence have shown remarkable improvement in protein yields from B. licheniformis expression systems, with up to 5-fold increases in protein production compared to single RBS constructs .

Methodology recommendations:

• The use of thermosensitive, self-replicable plasmids like pUB-MazF combined with integrative plasmids such as pUB'-EX1 creates stable recombinant B. licheniformis strains with multiple gene copies

• Optimization of culture media composition through response surface methodology (RSM) significantly enhances protein expression

• Experimental validation of multiple RBS constructs has demonstrated substantial increases in translation efficiency for various proteins in B. licheniformis

What are the optimal purification strategies for UPF0637 protein from B. licheniformis?

Purification of recombinant UPF0637 protein requires a multi-step approach to achieve high purity while maintaining structural integrity and biological activity:

Step-by-step methodology:

• Cell lysis and initial clarification:

  • Mechanical disruption (sonication or high-pressure homogenization) in a buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, and protease inhibitors

  • Centrifugation at 15,000 × g for 30 minutes at 4°C to remove cell debris

• Capture chromatography:

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

  • For native protein: Ion exchange chromatography using a strong anion exchanger (Q-Sepharose)

• Intermediate purification:

  • Size exclusion chromatography to separate protein aggregates and different oligomeric states

  • Recommend Superdex 75 or Superdex 200 columns based on protein size

• Polishing step:

  • Hydrophobic interaction chromatography or an additional ion exchange step

• Quality assessment:

  • SDS-PAGE analysis (>85% purity standard)

  • Western blot confirmation

  • Mass spectrometry verification

The purity of the final preparation should be confirmed using SDS-PAGE and should exceed 85% as this is the standard used for other recombinant B. licheniformis proteins . For functional studies, additional verification of proper folding using circular dichroism or fluorescence spectroscopy is recommended.

How can researchers optimize UPF0637 protein stability and storage conditions?

Maintaining protein stability is crucial for experimental reproducibility and functional studies:

Table 2: Storage Condition Optimization for Recombinant UPF0637 Protein

The shelf life of the recombinant protein is influenced by multiple factors including storage state, buffer ingredients, storage temperature, and the intrinsic stability of the protein itself. Generally, liquid formulations have a shelf life of approximately 6 months at -20°C/-80°C, while lyophilized forms can maintain stability for 12 months or longer at -20°C/-80°C .

Methodological considerations for stability assessment:

• Thermal shift assays to determine buffer conditions that maximize protein thermal stability

• Size exclusion chromatography to monitor aggregation over time

• Enzyme activity assays (if applicable) to confirm retention of functional properties

• Dynamic light scattering to detect early signs of aggregation

What experimental approaches can reveal the biological function of UPF0637 protein?

Given the uncharacterized nature of UPF0637 protein, a multi-faceted experimental approach is necessary:

• Genetic approaches:

  • Gene knockout studies using CRISPR-Cas9 or traditional homologous recombination

  • Complementation assays with the wild-type gene in knockout strains

  • Overexpression studies to identify gain-of-function phenotypes

• Biochemical characterization:

  • Substrate specificity screening using diverse compound libraries

  • Protein-protein interaction studies (pull-down assays, yeast two-hybrid)

  • Enzymatic activity assays based on predicted functions from structural similarities

• Structural biology approaches:

  • X-ray crystallography or cryo-EM to determine three-dimensional structure

  • NMR spectroscopy for dynamic structural information

  • In silico docking studies with potential substrates or binding partners

• Phenotypic analysis:

  • Comparison of wild-type and knockout strains under various stress conditions

  • Assessment of changes in antimicrobial peptide production

  • Evaluation of biofilm formation capabilities and cell morphology

Since B. licheniformis produces various antimicrobial substances including bacteriocins (1.4-20 kDa), non-ribosomally synthesized peptides and cyclic lipopeptides (0.8-42 kDa), and exopolysaccharides (>1000 kDa) , investigating potential connections between UPF0637 protein and these pathways could reveal functional insights.

How might UPF0637 protein contribute to B. licheniformis antimicrobial properties?

B. licheniformis is known for producing diverse antimicrobial compounds that act against various pathogens. While the direct role of UPF0637 protein in antimicrobial activity has not been established, several potential mechanisms warrant investigation:

• Regulatory role in antimicrobial peptide production:

  • The protein may function as a transcriptional regulator controlling expression of antimicrobial peptide genes

  • It could be involved in post-translational modification of antimicrobial peptides to enhance their activity

• Direct antimicrobial activity:

  • Some uncharacterized proteins have been found to possess direct antimicrobial properties

  • Size-exclusion chromatography followed by antimicrobial activity assays of purified fractions could determine if UPF0637 protein exhibits direct antimicrobial activity

• Role in secretion pathways:

  • The protein might participate in the secretion machinery for antimicrobial compounds

  • Analysis of secretome in wild-type versus knockout strains could reveal altered secretion patterns

B. licheniformis produces several antimicrobial agents with different mechanisms of action, including:

Table 3: Antimicrobial Compounds Produced by B. licheniformis and Their Properties

This extracellular protein from B. licheniformis D1 demonstrates significant biofilm inhibition with minimum inhibitory concentration (MIC) values of 1.6 µg/ml against C. albicans and 3.12 µg/ml against P. aeruginosa and B. pumilus . Investigating whether UPF0637 protein has similar activities or regulates the production of such compounds would be valuable.

How can chromosomal integration strategies improve UPF0637 protein expression?

For stable and high-level expression of UPF0637 protein, chromosomal integration offers significant advantages over plasmid-based systems:

Methodology for chromosomal integration of UPF0637 gene:

• Construction of integration cassettes:

  • Design integration cassette containing UPF0637 gene with strong promoter (P43 promoter has shown excellent results)

  • Include multiple ribosomal binding sites to enhance translation efficiency (5-6 RBSs can increase protein production up to 5-fold)

  • Flank with homologous regions targeting desired chromosomal location

• Transformation and selection approach:

  • Utilize the pUB-MazF/pUB'-EX1 system for efficient multi-copy integration

  • The pUB-MazF plasmid is thermosensitive and curable through MazF toxin expression

  • The pUB'-EX1 plasmid facilitates integration into the chromosome

• Verification of integration and copy number:

  • PCR verification of integration site

  • qPCR determination of gene copy number

  • Assessment of expression levels by Western blot or activity assays

The integration procedure follows a three-step process:

• Transform pUB-MazF into B. licheniformis and select for integration at elevated temperature (42°C)

• Transform pUB'-EX1 containing UPF0637 gene into cells harboring pUB-MazF

• Induce MazF expression with IPTG at 42°C, forcing pUB'-EX1 integration while curing pUB-MazF

This method has demonstrated remarkable success, yielding strains with multiple gene copies and stable expression over extended fermentation periods. For α-amylase expression, a strain with five gene copies produced 50,753 U/ml after 72 hours of cultivation, a 22-fold improvement over previous methods .

What strategies can optimize codon usage for enhanced UPF0637 protein expression?

Codon optimization significantly impacts recombinant protein expression levels, particularly in B. licheniformis:

Codon optimization methodology:

• Analysis of B. licheniformis codon usage bias:

  • Calculate codon adaptation index (CAI) for native UPF0637 gene

  • Identify rare codons that might limit translation efficiency

  • Analyze GC content and potential secondary structures in mRNA

• Design optimization strategies:

  • Replace rare codons with synonymous codons preferred by B. licheniformis

  • Eliminate potential mRNA secondary structures that might impede translation

  • Optimize 5' region of the coding sequence to enhance translation initiation

• Experimental validation:

  • Construct expression vectors with both native and codon-optimized genes

  • Compare expression levels using Western blot and activity assays

  • Evaluate mRNA levels using RT-qPCR to distinguish transcriptional from translational effects

Table 4: Impact of Optimization Strategies on Protein Expression

RT-qPCR analysis of gene transcription levels, coupled with polysome profiling, can determine whether increased protein production results from enhanced translation rather than increased transcription. This methodology has verified that multiple RBS constructs significantly improve translation efficiency without affecting transcription levels .

How does UPF0637 protein structure relate to its potential function?

While the specific structure of UPF0637 protein from B. licheniformis has not been fully characterized, computational analysis and structural predictions can provide valuable insights:

• Homology modeling approach:

  • Identify closest structural homologs in the Protein Data Bank

  • Generate models using software like SWISS-MODEL, Phyre2, or I-TASSER

  • Validate models through energy minimization and Ramachandran plot analysis

• Functional prediction from structural features:

  • Identify potential active sites or binding pockets

  • Analyze surface electrostatic potential for clues about molecular interactions

  • Examine conserved structural motifs shared with proteins of known function

• Experimental structure determination:

  • Express and purify UPF0637 protein for crystallization trials

  • Optimize buffer conditions to enhance protein stability and crystal formation

  • Collect X-ray diffraction data and solve structure using molecular replacement or experimental phasing

Based on sequence analysis of other characterized proteins from B. licheniformis, UPF0637 may share structural features with proteins involved in antimicrobial peptide production, secretion, or regulation. The protein might contain specific domains for RNA or protein interactions, suggesting a potential regulatory role.

What protein-protein interaction studies could reveal UPF0637 function?

Investigating protein-protein interactions (PPIs) represents a powerful approach to elucidate the function of uncharacterized proteins like UPF0637:

Methodological approaches for PPI studies:

• Affinity purification-mass spectrometry (AP-MS):

  • Express tagged UPF0637 protein in B. licheniformis

  • Perform pull-down assays under various growth conditions

  • Identify binding partners through mass spectrometry

  • Validate interactions using reciprocal pull-downs

• Bacterial two-hybrid system:

  • Screen for potential interaction partners using B. licheniformis genomic library

  • Validate positive interactions through secondary assays

  • Map interaction domains through truncation studies

• Cross-linking coupled with mass spectrometry:

  • Use chemical cross-linkers to capture transient interactions

  • Identify cross-linked peptides by mass spectrometry

  • Generate interaction network maps

• Co-immunoprecipitation studies:

  • Develop antibodies against UPF0637 protein

  • Perform co-IP experiments under various cellular conditions

  • Identify precipitated proteins by Western blot or mass spectrometry

Based on B. licheniformis biology, UPF0637 might interact with proteins involved in antimicrobial compound production, stress response, or regulatory networks. Investigating interactions with known components of these pathways could provide functional insights.