Recombinant Bacillus licheniformis UPF0754 membrane protein BLi01057/BL02871 (BLi01057, BL02871)

What is the UPF0754 membrane protein from Bacillus licheniformis?

The UPF0754 membrane protein from Bacillus licheniformis (strain DSM 13 / ATCC 14580) is a full-length protein of 376 amino acids encoded by the genes BLi01057 and BL02871 . This protein belongs to the UPF (Uncharacterized Protein Family) classification, specifically the UPF0754 group, indicating that its function has not been fully characterized experimentally . The protein is assigned the UniProt accession number Q65LU9 and has distinctive transmembrane regions that anchor it within the bacterial cell membrane . Based on its amino acid sequence and structural predictions, this membrane protein likely plays a role in cellular processes associated with the bacterial membrane, possibly in transport, signaling, or maintaining membrane integrity.

How stable is the recombinant UPF0754 membrane protein during laboratory storage and handling?

The recombinant UPF0754 membrane protein requires specific storage conditions to maintain stability. According to product specifications, the protein should be stored at -20°C, or at -80°C for extended storage periods . The protein is typically supplied in a Tris-based buffer containing 50% glycerol, which has been optimized for this specific protein to enhance stability . For working solutions, it is recommended to store aliquots at 4°C for no more than one week . Importantly, repeated freezing and thawing cycles should be avoided as they can lead to protein denaturation and loss of structural integrity . When handling the protein for experiments, maintaining a consistent temperature and minimizing exposure to proteases are essential practices to preserve its native conformation and activity.

What expression systems are most effective for producing recombinant UPF0754 membrane protein?

For recombinant membrane proteins like UPF0754, insect cell expression systems have proven particularly effective. Based on similar recombinant protein studies, the baculovirus expression vector system (BEVS) using Spodoptera frugiperda (Sf9) cells represents one of the most efficient platforms for producing membrane proteins with proper folding and post-translational modifications . The baculovirus approach involves generating recombinant baculovirus bacmid DNA carrying the target gene, transfecting insect cells, and then collecting progeny viruses through consecutive rounds of infection . This system is advantageous because insect cells provide a eukaryotic environment that supports complex protein folding while allowing for high expression levels driven by the strong polyhedrin promoter (PPH) . Alternative systems include E. coli-based expression, though these may require optimization of conditions to prevent inclusion body formation due to the hydrophobic nature of membrane proteins.

What purification strategies yield the highest purity for UPF0754 membrane protein?

Purification of membrane proteins like UPF0754 typically requires a multi-step approach. Based on standard protocols for similar proteins, an effective strategy would begin with cell lysis using detergent-based buffers that can solubilize membrane proteins while maintaining their native conformation . For tagged versions of the protein (such as His-Flag tagged constructs), immobilized metal affinity chromatography (IMAC) using Ni-NTA resin provides an excellent first purification step . This can be followed by size exclusion chromatography (SEC) to separate the target protein from aggregates and other contaminants based on molecular size. Ion exchange chromatography might serve as an additional purification step depending on the protein's isoelectric point. Western blot analysis using anti-Flag antibodies can confirm the identity and purity of the target protein, which typically appears as a band of approximately 38 kDa (close to the predicted molecular weight of 37.72 kDa) . Throughout the purification process, maintaining the protein in buffers containing appropriate detergents is crucial to prevent aggregation.

How can researchers optimize the secretory expression of UPF0754 membrane protein in insect cells?

Optimizing secretory expression of UPF0754 membrane protein in insect cells requires attention to several key factors. First, the incorporation of an appropriate signal peptide at the N-terminus of the protein sequence is essential to direct the protein to the secretory pathway . The codon optimization of the gene sequence for insect cell expression systems significantly improves translation efficiency, as demonstrated in similar recombinant protein studies . Infection parameters, including multiplicity of infection (MOI), timing of harvest (typically 72 hours post-infection when cytopathic effects become apparent), and culture conditions (27°C without CO2 exchange) must be carefully optimized .

The addition of tags (such as His and Flag tags) not only facilitates purification but can also enhance protein solubility and detection . To monitor secretion efficiency, researchers should analyze both cell lysates and culture supernatants by SDS-PAGE and Western blotting using appropriate antibodies (e.g., anti-Flag) . Successful secretion is confirmed when the target protein is detected in the culture supernatant, indicating that translation and secretion have occurred in the infected cells . Optimization of media composition, particularly the addition of components that support protein folding and secretion, can further enhance yields of functional secreted protein.

What methods are most suitable for determining the membrane topology of UPF0754 protein?

Determining the membrane topology of UPF0754 requires a combination of computational prediction and experimental validation approaches. Computational methods include transmembrane prediction algorithms (such as TMHMM, HMMTOP, or Phobius) which can identify potential membrane-spanning regions based on the amino acid sequence . The hydrophobic N-terminal (MYVFGIFAVMIAVGALIGAVTNH) and C-terminal (LGGIIGAVQAIFVILI) regions of UPF0754 are likely to form transmembrane domains .

For experimental validation, techniques such as cysteine scanning mutagenesis coupled with accessibility assays can determine which portions of the protein are exposed to the cytoplasm, periplasm, or embedded within the membrane. Protease protection assays using proteases that cannot penetrate the membrane can help identify exposed regions. Fluorescence techniques, including site-directed fluorescence labeling at termini or within loop regions, provide information about protein orientation. Epitope insertion followed by immunofluorescence microscopy without cell permeabilization can reveal which parts of the protein are accessible from the extracellular environment. Cryo-electron microscopy (cryo-EM) represents an advanced approach that can provide high-resolution structural information while the protein remains in a membrane-like environment, offering insights into both topology and tertiary structure.

How does one distinguish between properly folded and misfolded forms of the recombinant UPF0754 membrane protein?

Distinguishing between properly folded and misfolded forms of recombinant UPF0754 membrane protein requires multiple analytical approaches. Circular dichroism (CD) spectroscopy can assess secondary structure content, with properly folded membrane proteins typically showing characteristic α-helical signatures with minima at 208 and 222 nm. Thermal stability assays that monitor protein unfolding as a function of temperature can provide information about structural integrity, as properly folded proteins generally exhibit cooperative unfolding transitions.

Size exclusion chromatography profiles can identify aggregated (misfolded) versus monomeric or oligomeric (likely properly folded) species . Properly folded membrane proteins typically elute at volumes corresponding to their predicted molecular weight plus the detergent micelle size. Functional assays, although challenging for uncharacterized proteins like UPF0754, can indirectly indicate proper folding if the protein exhibits expected activities. Limited proteolysis experiments can also distinguish folded from misfolded states, as properly folded proteins typically show resistance to proteolytic digestion except at exposed loop regions. For tagged constructs, comparing the accessibility of tags in detergent-solubilized versus denatured conditions can provide insights into protein conformation.

What are the challenges and solutions for crystallizing UPF0754 membrane protein for X-ray crystallography?

Crystallizing membrane proteins like UPF0754 presents several significant challenges. The amphipathic nature of membrane proteins requires detergents to maintain solubility, but detergent micelles can interfere with crystal contacts. The conformational heterogeneity common in membrane proteins can hinder the formation of ordered crystals. Additionally, membrane proteins often have limited hydrophilic surfaces available for crystal contacts.

Effective solutions include screening a wide range of detergents to identify those that maintain protein stability while allowing crystallization. Lipidic cubic phase (LCP) crystallization methods often prove successful for membrane proteins by providing a membrane-mimetic environment. The use of antibody fragments (Fab or nanobody) as crystallization chaperones can increase the hydrophilic surface area available for crystal contacts. Protein engineering approaches, such as removing flexible regions or introducing thermostabilizing mutations, can enhance crystallizability. Construct optimization through systematic N and C-terminal truncations may identify more crystallization-prone variants. For particularly challenging targets, alternative structural biology approaches such as cryo-EM may be considered, as this technique has advanced significantly for membrane proteins and does not require crystallization.

How can researchers determine the physiological role of UPF0754 membrane protein in Bacillus licheniformis?

Determining the physiological role of UPF0754 membrane protein requires a multi-faceted approach. Gene knockout or knockdown studies in B. licheniformis using CRISPR-Cas9 or antisense RNA can reveal phenotypic changes associated with protein absence. These changes should be assessed under various growth conditions to identify stress-specific functions. Complementation studies, where the wild-type gene is reintroduced into knockout strains, can confirm that observed phenotypes are directly related to the absence of UPF0754.

Transcriptomic and proteomic analyses comparing wild-type and UPF0754-deficient strains can identify dysregulated pathways, providing clues about the protein's functional network. Co-immunoprecipitation followed by mass spectrometry can identify interaction partners, placing UPF0754 within specific cellular pathways. Bacterial two-hybrid or pull-down assays can validate these interactions. Localization studies using fluorescently tagged UPF0754 can reveal subcellular distribution patterns that may suggest function. Comparative genomics and phylogenetic analyses across bacterial species can identify conservation patterns and potential functional homologs. For membrane proteins, transport assays using reconstituted proteoliposomes might determine if UPF0754 functions as a transporter for specific substrates.

What bioinformatic approaches can predict potential functions of UPF0754 protein?

Bioinformatic approaches provide valuable insights into potential functions of uncharacterized proteins like UPF0754. Sequence similarity searches using tools like BLAST against characterized proteins may identify functional homologs . Protein family classification using Pfam, InterPro, or CATH can place UPF0754 within known functional groups. Structural prediction using AlphaFold2 or RoseTTAFold can generate high-confidence models that may reveal structural similarities to proteins with known functions, even in the absence of significant sequence similarity.

Conserved domain analyses can identify functional motifs within the protein sequence . The presence of leucine-rich regions in UPF0754 suggests potential protein-protein interaction domains. Genomic context analysis examining the organization of genes surrounding BLi01057/BL02871 may reveal operons or functional gene clusters. Phylogenetic profiling can identify proteins with similar evolutionary patterns across species, suggesting functional relationships. Protein-protein interaction predictions using tools like STRING can generate hypothetical functional networks. Molecular docking simulations with potential ligands or substrates can test binding hypotheses. Transmembrane topology predictions using TMHMM or Phobius can provide insights into membrane orientation relevant to function .

What experimental design is optimal for identifying potential binding partners of UPF0754 membrane protein?

An optimal experimental design for identifying binding partners of UPF0754 membrane protein would employ complementary in vivo and in vitro approaches. For in vivo detection, proximity-dependent biotin identification (BioID) or APEX2 proximity labeling represents an excellent approach. The UPF0754 protein is fused to a promiscuous biotin ligase, expressed in B. licheniformis, and nearby proteins become biotinylated. These proteins are then purified using streptavidin and identified by mass spectrometry.

Affinity purification using tagged UPF0754 (as described in the research) followed by mass spectrometry (AP-MS) can isolate stable interacting partners . Crosslinking mass spectrometry (XL-MS) utilizes chemical crosslinkers to capture transient interactions before purification and identification. For membrane protein-specific interactions, split-ubiquitin membrane yeast two-hybrid systems are particularly suitable. Co-immunoprecipitation experiments using antibodies against UPF0754 can pull down interacting proteins from native contexts .

In vitro approaches include reconstitution of purified UPF0754 into liposomes or nanodiscs followed by pull-down assays with cellular extracts. Surface plasmon resonance (SPR) or microscale thermophoresis (MST) can validate and quantify specific protein-protein interactions. To ensure biological relevance, validation of identified interactions should be performed through techniques such as co-localization studies, FRET analysis, or functional assays examining the effects of disrupting specific interactions.

How can researchers address poor expression yields of UPF0754 membrane protein?

Poor expression yields of UPF0754 membrane protein can be addressed through systematic optimization of expression conditions. Codon optimization for the expression host is crucial, as demonstrated in studies using insect cell systems where codon-optimized sequences significantly improved protein yields . For baculovirus expression systems, optimizing the multiplicity of infection (MOI) and harvest time point (typically 72 hours post-infection) can substantially impact yields .

Expression vector selection is critical; vectors with strong promoters like the polyhedrin promoter (PPH) in baculovirus systems drive high-level expression . Adding solubility-enhancing fusion tags such as SUMO, MBP, or Thioredoxin can improve expression of challenging membrane proteins. Temperature modulation during expression (often lowering to 27°C for insect cells or 16-18°C for bacterial systems) can enhance proper folding .

Media supplementation with chemical chaperones like glycerol, arginine, or specific detergents can prevent aggregation during expression. For toxic membrane proteins, using tightly regulated inducible systems prevents premature expression that may hinder cell growth. Host strain selection is also important; certain strains of E. coli (C41(DE3), C43(DE3)) are engineered specifically for membrane protein expression. Monitoring protein expression through methods like Western blotting of both cell lysates and culture supernatants helps identify the optimal expression conditions .

What are the common pitfalls in purifying UPF0754 membrane protein and how can they be overcome?

Common pitfalls in purifying UPF0754 membrane protein include protein aggregation, poor solubilization, low binding efficiency to affinity resins, and loss of native structure. To overcome aggregation issues, selecting appropriate detergents is crucial, with mild non-ionic detergents like DDM, LMNG, or digitonin often proving effective for maintaining membrane protein solubility without denaturation. Screening multiple detergents at various concentrations should be performed to identify optimal conditions.

For efficient solubilization, the detergent concentration should be well above its critical micelle concentration (CMC), and solubilization should be performed at 4°C with gentle agitation. Adding glycerol (10-20%) and specific lipids to purification buffers can enhance protein stability. When using affinity chromatography with tagged constructs (His-tag, Flag-tag), optimizing imidazole concentrations in wash buffers prevents non-specific binding while maximizing target protein retention .

Size exclusion chromatography should be performed immediately after affinity purification to separate aggregates from properly folded protein . Throughout purification, protein stability should be monitored using techniques like dynamic light scattering or thermal shift assays. For difficult-to-purify membrane proteins, alternative approaches such as native purification without detergent extraction using styrene-maleic acid lipid particles (SMALPs) or nanodiscs may preserve native lipid interactions and improve stability.

How can researchers validate that recombinant UPF0754 maintains its native conformation after purification?

Validating that recombinant UPF0754 maintains its native conformation after purification requires multiple complementary approaches. Circular dichroism (CD) spectroscopy can assess secondary structure content, with α-helical membrane proteins exhibiting characteristic spectra with minima at 208 and 222 nm. Thermal stability measurements using differential scanning calorimetry (DSC) or thermal shift assays can provide information on protein folding, as properly folded proteins typically show cooperative unfolding transitions.

Fluorescence spectroscopy utilizing intrinsic tryptophan fluorescence can detect conformational changes, as the emission spectra of tryptophan residues shift depending on their local environment. Size exclusion chromatography profiles should show monodisperse peaks corresponding to the expected molecular weight plus detergent micelle size, without significant aggregate formation . Limited proteolysis experiments can assess structural integrity, as properly folded proteins typically show resistance to proteolytic digestion except at exposed loop regions.

For functional validation, ligand binding assays (if ligands are known) can confirm that binding sites remain intact. Electron microscopy (negative stain or cryo-EM) can provide direct visualization of protein particles, confirming proper folding and the absence of aggregation. For tagged proteins, tag accessibility assays comparing native versus denatured conditions can indicate whether the protein maintains its expected conformation .

How can UPF0754 membrane protein be utilized in structural biology studies of bacterial membrane proteins?

UPF0754 membrane protein can serve as a valuable model system for structural biology studies of bacterial membrane proteins. As an uncharacterized protein family member, structural determination of UPF0754 would contribute significantly to understanding novel membrane protein folds and potentially identify new structural motifs. For structural studies, the protein can be expressed with various constructs including truncations and fusion proteins to identify versions with enhanced stability and crystallizability .

Advanced techniques such as lipidic cubic phase (LCP) crystallization are particularly suitable for membrane proteins like UPF0754. Cryo-electron microscopy (cryo-EM) represents an increasingly powerful approach that can provide high-resolution structures without the need for crystallization, especially when combined with innovative approaches like membrane scaffold proteins or nanodiscs. Solid-state NMR studies of uniformly or selectively labeled UPF0754 can provide detailed information about dynamics and conformational changes within the membrane environment.

The protein can also serve as a platform for developing improved methods for membrane protein solubilization, such as novel detergents, amphipols, or nanodiscs. Comparative structural studies between UPF0754 from different bacterial species could provide evolutionary insights into membrane protein structural conservation. Integration of structural data with functional studies would contribute to our understanding of structure-function relationships in membrane proteins of unknown function.

What strategies can improve the stability of UPF0754 membrane protein for long-term functional studies?

Improving the stability of UPF0754 membrane protein for long-term functional studies requires strategies addressing both intrinsic and environment-dependent factors. Protein engineering approaches can enhance intrinsic stability through introducing disulfide bonds at strategic positions, performing alanine scanning to identify and mutate destabilizing residues, or using directed evolution to select for thermostable variants. Consensus design, which incorporates the most common amino acids from sequence alignments of homologous proteins, can also yield more stable variants.

Environment optimization is equally important. Detergent screening to identify those that best mimic the native membrane environment is essential . Supplementing buffers with specific lipids that interact with UPF0754 can significantly enhance stability. The addition of cholesterol hemisuccinate (CHS) often improves membrane protein stability. Buffer optimization should include testing various pH values, salt concentrations, and additives such as glycerol (10-20%) .

For long-term storage, lyophilization in the presence of appropriate stabilizers may be considered. Reconstitution into more native-like membrane environments such as nanodiscs, liposomes, or amphipols often provides superior stability compared to detergent micelles . Covalent modification approaches, such as PEGylation of solvent-exposed lysine residues, can improve solubility without affecting function. Finally, co-expression or addition of stabilizing binding partners (antibody fragments, ligands, or lipids) can lock the protein in stable conformations.

How can computational modeling enhance our understanding of UPF0754 membrane protein dynamics?

Computational modeling can significantly enhance our understanding of UPF0754 membrane protein dynamics through multiple approaches. Homology modeling using AlphaFold2 or RoseTTAFold can generate high-confidence structural models even with limited sequence similarity to known structures. These models can provide initial insights into the protein's architecture and potential functional sites. Molecular dynamics (MD) simulations of UPF0754 embedded in a lipid bilayer can reveal dynamic behaviors including conformational changes, lipid interactions, and potential substrate binding pathways.

Course-grained MD simulations allow for longer timescale events to be observed, potentially capturing large-scale movements relevant to function. Normal mode analysis can identify intrinsic flexibility and potential functional motions with minimal computational cost. Continuum electrostatics calculations can map the electrostatic potential on the protein surface, highlighting potential binding sites for charged molecules or proteins.

Molecular docking combined with MD can predict binding modes of potential ligands or interaction partners. Free energy calculations can estimate binding affinities and identify key residues contributing to interactions. Markov state modeling can identify metastable states and transition pathways between conformations. Integration of computational predictions with experimental data (such as site-directed mutagenesis results or spectroscopic measurements) in an iterative fashion can progressively refine our understanding of UPF0754 dynamics and function.

What are the current knowledge gaps regarding UPF0754 membrane protein research?

Significant knowledge gaps persist in UPF0754 membrane protein research despite advances in recombinant protein technology. The physiological function of this protein family remains largely uncharacterized, with limited understanding of its role in Bacillus licheniformis cellular processes . The three-dimensional structure of UPF0754 has not been experimentally determined, hampering structure-based functional predictions and targeted drug design efforts. The specific ligands, substrates, or binding partners that interact with UPF0754 in vivo have not been definitively identified, leaving its position in cellular signaling or metabolic networks unclear .

The regulatory mechanisms controlling UPF0754 expression under different environmental conditions remain unexplored. Whether UPF0754 functions as a monomer or forms homo/hetero-oligomeric complexes is unknown. The post-translational modifications that might regulate UPF0754 activity have not been characterized. Conservation analysis across bacterial species is incomplete, limiting evolutionary insights into functional importance. Comprehensive mutagenesis studies mapping critical functional residues have not been reported. Additionally, the potential of UPF0754 as a target for antimicrobial development or biotechnological applications remains largely unexplored despite the increasing relevance of Bacillus licheniformis in industrial processes.

What future research directions will advance our understanding of UPF0754 membrane protein?

Future research directions to advance our understanding of UPF0754 membrane protein should focus on integrative approaches spanning multiple disciplines. High-resolution structural determination using cryo-electron microscopy or X-ray crystallography would provide critical insights into protein architecture and potential functional sites. Comprehensive protein-protein interaction studies using techniques like BioID, crosslinking mass spectrometry, or membrane yeast two-hybrid screens would identify interaction partners and place UPF0754 within cellular networks .

Systematic gene knockout studies in Bacillus licheniformis coupled with phenotypic characterization under various environmental conditions would reveal functional importance in bacterial physiology. Multi-omics approaches (transcriptomics, proteomics, metabolomics) comparing wild-type and UPF0754-deficient strains could identify affected pathways suggesting function. Development of functional assays based on bioinformatic predictions to test hypothesized activities (such as transport, enzymatic activity, or signaling) would provide direct evidence of function.