Recombinant UPF0754 membrane protein BA_0862/GBAA_0862/BAS0819 (BA_0862, GBAA_0862, BAS0819)

What is UPF0754 membrane protein BA_0862/GBAA_0862/BAS0819?

UPF0754 membrane protein BA_0862/GBAA_0862/BAS0819 is a membrane-associated protein from Bacillus anthracis with UniProt accession number Q81UK6. The protein consists of 378 amino acids and represents a member of the uncharacterized protein family (UPF) 0754. The designations BA_0862, GBAA_0862, and BAS0819 represent ordered locus names for the same gene in different B. anthracis strains . As a membrane protein, it contains multiple transmembrane domains that anchor it within the bacterial cell membrane, suggesting potential roles in transport, signaling, or membrane integrity maintenance.

What expression systems are recommended for producing this recombinant protein?

Multiple expression systems can be utilized for the production of UPF0754 membrane protein, each with distinct advantages for different research applications:

E. coli and yeast systems typically offer the best yields with shorter turnaround times, making them suitable for initial characterization studies . For more complex functional analyses requiring proper post-translational modifications, insect or mammalian expression systems may be preferable despite their higher cost and lower yield.

What are the optimal storage conditions for maintaining protein stability?

For optimal stability of recombinant UPF0754 membrane protein:

• Store at -20°C for routine storage or -80°C for extended preservation

• Utilize a Tris-based buffer containing 50% glycerol as a cryoprotectant

• Maintain working aliquots at 4°C for up to one week

• Avoid repeated freeze-thaw cycles that can compromise protein integrity

Creating single-use aliquots during initial preparation is strongly recommended to minimize the need for multiple freeze-thaw events that can lead to protein denaturation and loss of function.

What methodological approaches optimize membrane protein purification?

Purifying membrane proteins like UPF0754 requires specialized techniques to maintain native structure and function:

• Solubilization optimization:

  • Systematic screening of detergent types (DDM, LDAO, CHAPS) at varying concentrations

  • Assessment of critical detergent:protein ratios

  • Evaluation of mixed micelle systems with lipids or amphipols

• Chromatographic strategy development:

  • Initial capture using affinity chromatography (His-tag, GST, etc.)

  • Secondary purification via ion exchange chromatography

  • Final polishing using size exclusion chromatography

  • Assessing detergent exchange during purification

• Stability enhancement techniques:

  • Addition of specific lipids (phosphatidylcholine, cardiolipin)

  • Buffer optimization (pH gradients, salt concentration screens)

  • Inclusion of stabilizing ligands or inhibitors

  • Temperature optimization during purification steps

• Alternative approaches:

  • Nanodisc reconstitution for detergent-free systems

  • Styrene maleic acid lipid particles (SMALPs) extraction

  • Saposin-lipoprotein nanoparticle systems

Throughout purification, progressive quality assessment via SDS-PAGE, Western blotting, and functional assays should guide protocol refinement for optimal results.

How do chaperone systems influence membrane protein folding and quality control?

Membrane proteins undergo complex folding processes involving specialized chaperone systems:

• Co-translational folding mechanisms:

  • SRP (Signal Recognition Particle) targeting to the membrane

  • SecYEG translocon-mediated insertion

  • YidC-assisted folding of transmembrane domains

  • Ribosome-associated chaperone interactions

• Post-translational quality control:

  • Hsp70/Hsp40 chaperone systems recognize misfolded membrane proteins and facilitate their interaction with E3 ubiquitin ligases

  • Calnexin/calreticulin cycle for glycosylated membrane proteins

  • Specialized membrane protein chaperones (e.g., TRAM, TRAP complexes)

Experimental data indicates that cytoplasmic Hsp70/Hsp40 chaperones directly facilitate ubiquitination of membrane proteins in vivo and in vitro, suggesting an active role in quality control rather than simply preventing aggregation .

What mechanisms govern membrane protein degradation pathways?

Understanding UPF0754 membrane protein degradation requires investigation of established membrane protein degradation pathways:

• ERAD pathway components:

  • Recognition by Hsp70/Hsp40 chaperones that facilitate interaction with E3 ubiquitin ligases

  • Ubiquitination by specific E3 ligases (such as Doa10p and Hrd1p in yeast)

  • Extraction from the membrane by the Cdc48/p97 AAA ATPase complex

  • Delivery to the 26S proteasome for degradation

• Experimental approaches:

  • In vitro ubiquitination assays using microsomes containing the protein of interest

  • Reconstituted systems with purified components

  • Cross-linking studies to capture transient interactions

  • Protease protection assays to assess membrane extraction

Research has demonstrated that ubiquitinated membrane proteins can be fully extracted from the membrane in an ATP-dependent manner by the Cdc48 complex before delivery to the proteasome .

What techniques are recommended for membrane protein topology determination?

Determining the topology of UPF0754 membrane protein requires complementary approaches:

• Computational methods:

  • Transmembrane prediction algorithms (TMHMM, Phobius)

  • Hydropathy plot analysis (Kyte-Doolittle)

  • Topology prediction servers (TOPCONS, MEMSAT)

• Biochemical approaches:

  • Cysteine scanning mutagenesis with thiol-reactive probes

  • Protease protection assays for loop identification

  • Glycosylation site mapping for lumenal domain identification

  • Epitope insertion and accessibility analysis

• Biophysical techniques:

  • Fluorescence spectroscopy with environment-sensitive probes

  • FRET analysis between labeled domains

  • Electron paramagnetic resonance (EPR) for distance measurements

  • Hydrogen-deuterium exchange mass spectrometry

Combining multiple methodologies provides cross-validation and resolves ambiguities in membrane topology models.

How can researchers investigate protein-protein interactions involving membrane proteins?

Studying UPF0754 membrane protein interactions requires specialized techniques:

• In vivo approaches:

  • Split-ubiquitin yeast two-hybrid systems (specialized for membrane proteins)

  • Bimolecular fluorescence complementation (BiFC)

  • Proximity labeling methods (BioID, APEX)

  • FRET/BRET analysis in live cells

• In vitro methods:

  • Co-immunoprecipitation with membrane-compatible detergents

  • Surface plasmon resonance (SPR) with protein reconstituted in nanodiscs

  • Microscale thermophoresis (MST) for interaction affinity

  • Chemical cross-linking coupled with mass spectrometry

Data from search result indicates that cross-linking studies can effectively capture membrane protein interactions with degradation machinery components, as demonstrated by the DSP and DTSSP cross-linker experiments that identified Doa10p interaction with a membrane protein substrate .

What pathways govern membrane protein trafficking in bacteria?

Bacterial membrane protein trafficking involves several pathways that can be investigated for UPF0754:

• Secretion mechanisms:

  • Sec-dependent pathway utilizing the SecYEG translocon

  • Twin-arginine translocation (Tat) pathway for folded proteins

  • Type I-VI secretion systems for specific protein classes

• Determinants of localization:

  • Signal peptide sequences (N-terminal, internal, C-terminal)

  • Transmembrane domain characteristics

  • Specific targeting motifs in cytoplasmic domains

• Methodological approaches:

  • Fluorescent protein fusions for localization tracking

  • Immunoelectron microscopy for precise subcellular localization

  • Subcellular fractionation and immunoblotting

  • Protease accessibility assays

Systematic mutation of potential targeting sequences can identify the specific determinants guiding UPF0754 membrane protein localization in B. anthracis.

How do transmembrane domains influence protein sorting and trafficking?

Transmembrane domains play crucial roles in membrane protein trafficking, as evidenced by research on plant vacuolar sorting:

• Domain-specific functions:

  • Transmembrane domains can contain specific sorting signals directing proteins to different cellular compartments

  • The transmembrane domain of vacuolar sorting receptors directs proteins via the Golgi to lytic vacuole prevacuolar compartments

  • Cytoplasmic tail sequences can override transmembrane domain signals, creating multi-level sorting mechanisms

• Experimental approaches:

  • Domain swapping experiments between proteins with different localizations

  • Systematic mutagenesis of transmembrane region residues

  • Tracking trafficking using glycosylation status as Golgi transit markers

  • Colocalization studies with compartment-specific markers

Research on plant vacuolar proteins demonstrated that alpha-TIP cytoplasmic tail prevented traffic through the Golgi and redirected proteins to alternative organelles, illustrating how specific domains can fundamentally alter trafficking pathways .

What experimental approaches can elucidate the biological function of UPF0754 membrane protein?

Determining the function of uncharacterized membrane proteins requires multifaceted approaches:

• Genetic manipulation:

  • Gene deletion/knockout studies with phenotypic analysis

  • Complementation assays to confirm specificity

  • Conditional expression systems for essential genes

  • Site-directed mutagenesis of conserved residues

• Physiological assessment:

  • Growth curve analysis under various conditions

  • Stress response profiling (pH, temperature, oxidative)

  • Membrane permeability and integrity assays

  • Metabolite transport studies if transport function is suspected

• Structural insights to function:

  • Conserved domain identification through bioinformatics

  • Structural modeling and comparison to characterized proteins

  • Binding pocket identification and ligand docking studies

  • Structure-guided mutagenesis of potential functional sites

• Systems biology approaches:

  • Transcriptomic analysis of knockout/overexpression strains

  • Metabolomic profiling to identify metabolic impacts

  • Protein-protein interaction network mapping

  • Comparative genomics across related bacterial species

Integrating multiple lines of evidence provides robust functional characterization even for proteins with no initial functional annotation.

How do detergent selection and membrane mimetics impact experimental outcomes?

The choice of detergent and membrane mimetic system critically affects membrane protein research:

For UPF0754 membrane protein, systematic screening of these systems should evaluate:

• Protein stability through thermal denaturation assays

• Functional activity preservation if assays are available

• Compatibility with downstream analytical techniques

• Scale-up potential for structural biology applications

What considerations are important when designing constructs for membrane protein studies?

Construct design significantly impacts successful membrane protein research:

• Terminal modifications:

  • Affinity tag positioning (N-terminal, C-terminal, internal)

  • Cleavable vs. non-cleavable tags

  • Fluorescent protein fusion locations

  • Signal sequence retention or modification

• Domain engineering:

  • Truncation of flexible termini to improve crystallization

  • Domain isolation for partial structure determination

  • Creation of chimeric constructs with well-folding domains

  • Thermostabilizing mutations based on homology models

• Expression optimization:

  • Codon optimization for expression host

  • Fusion partners to enhance folding (MBP, SUMO, Mistic)

  • Sequence modifications to remove internal restriction sites

  • Incorporation of purification-enhancing elements

Construct libraries with systematic variations allow empirical determination of optimal designs for different experimental applications with UPF0754 membrane protein.