Recombinant Bacillus clausii UPF0754 membrane protein ABC1518 (ABC1518)

What is the Bacillus clausii UPF0754 membrane protein ABC1518?

Recombinant Bacillus clausii UPF0754 membrane protein ABC1518 (ABC1518) is a full-length protein (379 amino acids) derived from the probiotic bacterium Bacillus clausii. It belongs to the UPF0754 family of membrane proteins with a UniProt ID of Q5WHV2 . This protein can be expressed recombinantly in E. coli with an N-terminal His-tag for purification and research purposes. The biological function of this specific membrane protein remains under investigation, though it appears to be involved in bacterial membrane processes based on its sequence and structural predictions.

What is the complete amino acid sequence of the ABC1518 protein?

The full amino acid sequence of the Bacillus clausii UPF0754 membrane protein ABC1518 consists of 379 amino acids as follows:

MHWIWLVLLLAVVGAIVGAATNALAIRMLFRPHRAYSIGKWQLPFTPGLLPRRQKELAVQ LGNIVANHLLTAEGLGKKFGSTAFAAELTNWLKKQLASWLRSERTVESILKPLFQADIGR EHLVVQSKSWLKDRLKRYLQKNKEVPIKSVVPQELQDRLTDWLPEASALLLKRATAYIDS EEGEQRIGAMVRQFLTTKGKVGSMVSMFFSADKLTEYVLPEIKKFLHDEQTKETVQSLLQ TEWHRMLNRPLASFQAENYVDQFVDKAAEELEGKIPVLNWYNAPLSTWTTPYAEPLVERG VPVIVGMVTVYMEQHIADILSKLRLEEVIEEQVASFSMAHLEKLIMNITRRELHMITLLG GLIGGIVGLIQAVIVHFFY

Researchers should note the presence of hydrophobic segments that suggest transmembrane domains, which is consistent with its classification as a membrane protein.

How is the protein typically supplied for research applications?

For research applications, the recombinant ABC1518 protein is typically supplied as a lyophilized powder with purity greater than 90% as determined by SDS-PAGE . The storage buffer usually consists of Tris/PBS-based buffer with 6% trehalose at pH 8.0. For reconstitution, researchers should briefly centrifuge the vial before opening and reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 5-50% (commonly 50%) is recommended for long-term storage at -20°C/-80°C .

What expression systems are optimal for producing recombinant ABC1518 protein?

E. coli is the primary expression system used for recombinant production of the ABC1518 protein with an N-terminal His-tag . When designing an expression experiment, researchers should consider:

• Selection of appropriate E. coli strain (BL21(DE3) is commonly used for membrane proteins)

• Optimization of induction conditions (IPTG concentration, temperature, and duration)

• Codon optimization for E. coli if expressing the full-length sequence

• Inclusion of chaperones to assist proper folding of membrane proteins

• Use of detergents for extraction from membranes

• Purification strategy utilizing the His-tag for affinity chromatography

Alternative expression systems like Bacillus subtilis might be explored for improved folding of proteins from related Bacillus species.

How should experiments be designed to study ABC1518 protein function?

When designing experiments to investigate the function of ABC1518 protein, consider the following methodological approach:

Table 1: Experimental Design Framework for ABC1518 Functional Analysis

This experimental framework provides a systematic approach to characterizing ABC1518 function while incorporating appropriate controls to validate findings.

What are the recommended storage and handling procedures for optimal protein stability?

To maintain optimal stability of recombinant ABC1518 protein, researchers should follow these evidence-based protocols:

• Store lyophilized protein at -20°C/-80°C upon receipt

• Aliquot reconstituted protein to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

• For long-term storage, add glycerol to a final concentration of 50%

• Avoid repeated freeze-thaw cycles as they can compromise protein integrity

When handling the protein, ensure sterile conditions and work quickly to minimize exposure to room temperature. Proper reconstitution in the recommended buffer is critical for maintaining structural integrity and function.

How can ABC1518 be studied in the context of bacterial membrane biology?

The ABC1518 membrane protein presents unique opportunities for studying bacterial membrane biology. Advanced research approaches include:

• Membrane integration studies: Using proteoliposomes to study protein integration and orientation within lipid bilayers

• Biophysical characterization: Employing circular dichroism (CD) spectroscopy to determine secondary structure elements, particularly alpha-helical content typical of transmembrane domains

• Transport assays: If the protein functions in transport, develop assays using fluorescent or radioactive substrates to measure transport activity

• Topological mapping: Using cysteine-scanning mutagenesis combined with accessibility agents to map membrane topology

• Computational modeling: Utilizing homology modeling and molecular dynamics simulations to predict structure and dynamics

These approaches can provide comprehensive insights into the protein's role in bacterial membrane biology and potential implications for B. clausii survival and probiotic functions.

What techniques can elucidate structure-function relationships of the ABC1518 protein?

Understanding structure-function relationships requires integrating multiple experimental approaches:

Table 2: Structure-Function Analysis Techniques for ABC1518

These techniques, when used in combination, provide comprehensive insights into how specific structural elements contribute to protein function, potentially revealing mechanistic details of ABC1518's role in B. clausii.

How does ABC1518 relate to the antimicrobial and probiotic properties of B. clausii?

B. clausii is known for its probiotic properties, including antimicrobial activity, enhancement of barrier function, and immunomodulatory effects . While the specific role of ABC1518 in these functions is not explicitly described in the available research, membrane proteins often play crucial roles in:

• Antibiotic resistance: B. clausii contains chromosomally-encoded resistance genes for β-lactams, macrolides, aminoglycosides, and chloramphenicol

• Bacterial communication and competition: Potentially involved in bacteriocin secretion, as B. clausii produces peptides toxic to other bacterial species

• Adhesion to host tissues: Membrane proteins often mediate adhesion, which is critical for probiotic colonization

• Stress response: Contributing to acid and bile tolerance, which enables survival in the gastrointestinal tract

Research investigating ABC1518 knockout mutants could reveal its specific contributions to these probiotic properties, particularly if phenotypes related to antimicrobial activity or stress tolerance are observed.

What biosafety considerations should researchers observe when working with recombinant B. clausii proteins?

While working with recombinant proteins from B. clausii, researchers should be aware of potential biosafety considerations:

• Potential pathogenicity: Though B. clausii is generally considered safe, there are documented cases of prolonged bacteraemia (mean duration 64 days, range 14-93 days) associated with B. clausii probiotic use, even in an immunocompetent child

• Antibiotic resistance: B. clausii harbors chromosomally-encoded resistance genes to multiple antibiotics , which necessitates proper containment practices

• Laboratory containment: Standard Biosafety Level 1 (BSL-1) practices are typically sufficient for recombinant protein work, but institutional guidelines should be followed

• Waste disposal: Proper decontamination of materials coming into contact with the protein

Researchers should conduct a thorough risk assessment and follow institutional biosafety committee recommendations before initiating work with this protein.

How should researchers address potential contamination issues in experimental design?

When designing experiments with recombinant proteins, contamination control is critical for reliable results. Implement these strategies:

• Rigorous controls: Include positive and negative controls in all experiments

• Sterile technique: Use aseptic methods during protein handling

• Quality control testing: Regularly test protein preparations for microbial contamination

• Endotoxin testing: Check for endotoxin contamination that could confound immunological studies

• Data validation: Implement internal validation protocols to identify anomalous results

A comprehensive data table design approach is crucial for tracking potential contamination variables:

Table 3: Data Table Design for Contamination Monitoring

This structured approach allows for systematic monitoring of potential contamination and its effects on experimental outcomes .

What methods are most effective for studying ABC1518 interactions with other bacterial proteins?

To study protein-protein interactions involving ABC1518, researchers should consider these methodological approaches:

• Co-immunoprecipitation (Co-IP): Using antibodies against the His-tag to pull down ABC1518 and its interacting partners

• Bacterial two-hybrid systems: Modified for membrane proteins to identify potential interactors

• Chemical cross-linking coupled with mass spectrometry: To capture transient interactions within the membrane environment

• Förster resonance energy transfer (FRET): For studying interactions in living cells

• Surface plasmon resonance (SPR): For quantitative binding kinetics with purified interaction partners

Each method has specific advantages and limitations for membrane protein interaction studies, and combining multiple approaches provides the most robust evidence for genuine interactions.

How can researchers investigate the relationship between ABC1518 and antibiotic resistance mechanisms?

B. clausii strains possess chromosomally-encoded antibiotic resistance genes . To investigate potential roles of ABC1518 in antibiotic resistance:

• Gene knockout studies: Create ABC1518 deletion mutants and assess changes in antibiotic susceptibility profiles

• Overexpression analysis: Express ABC1518 in heterologous hosts and measure changes in antibiotic resistance

• Site-directed mutagenesis: Identify critical residues by creating point mutations and assessing functional changes

• Transport assays: If ABC1518 functions as a transporter, measure efflux/influx of antibiotics

• Transcriptional response: Monitor expression of ABC1518 under antibiotic stress conditions

Research findings could be organized using this experimental framework:

Table 4: Antibiotic Susceptibility Analysis Framework

This approach would systematically evaluate ABC1518's potential role in the known antibiotic resistance mechanisms of B. clausii.

How does ABC1518 compare with homologous proteins in other probiotic bacteria?

Comparative analysis of ABC1518 with homologous proteins in other bacteria can provide evolutionary and functional insights:

• Sequence alignment: Perform multiple sequence alignment with homologs from related Bacillus species and other probiotic bacteria

• Phylogenetic analysis: Construct phylogenetic trees to understand evolutionary relationships

• Domain conservation: Identify conserved functional domains and motifs

• Positive selection analysis: Calculate dN/dS ratios to identify positions under selective pressure

• Structural comparison: Where structures are available, compare structural features that may indicate functional conservation

What bioinformatic approaches can predict the functional role of ABC1518?

Advanced bioinformatic techniques can provide valuable predictions about ABC1518 function:

• Gene neighborhood analysis: Examine genomic context for clues about functional relationships

• Protein family classification: Identify membership in known protein families and superfamilies

• Transmembrane topology prediction: Identify membrane-spanning regions and orientation

• Ligand-binding site prediction: Identify potential binding pockets and substrates

• Molecular docking simulations: Predict interactions with potential ligands

• Co-expression network analysis: Identify genes with correlated expression patterns

These computational approaches can guide experimental design by generating testable hypotheses about ABC1518 function in B. clausii membranes.