Recombinant Staphylococcus haemolyticus UPF0754 membrane protein SH1116 (SH1116)

What expression systems are most effective for producing recombinant SH1116 protein?

For recombinant expression of SH1116, E. coli-based systems have proven effective, as documented in the production of His-tagged SH1116 . When designing expression systems for membrane proteins like SH1116, several methodological considerations are crucial:

• Vector selection: Vectors with tightly controlled promoters (such as T7) allow for regulated expression

• Host strain optimization: BL21(DE3) or C41/C43 strains specifically engineered for membrane protein expression are recommended

• Induction conditions: Lower temperatures (16-25°C) and reduced inducer concentrations often improve proper folding

• Media supplementation: Addition of glycerol (0.5-1%) can enhance membrane protein stability

The choice between prokaryotic and eukaryotic expression systems depends on research goals, with E. coli offering simplicity and high yields but potentially lacking post-translational modifications that might be relevant for functional studies .

What are the optimal purification protocols for maintaining SH1116 structural integrity?

Purification of SH1116 requires specialized approaches due to its membrane protein nature:

• Solubilization: Carefully selected detergents (DDM, LDAO, or Triton X-100) at concentrations just above their critical micelle concentration (CMC)

• Affinity chromatography: Immobilized metal affinity chromatography (IMAC) using the His-tag for initial capture

• Buffer composition: Inclusion of stabilizing agents such as glycerol (6%) in Tris/PBS-based buffers (pH 8.0)

• Storage: Lyophilization or storage in solution with 50% glycerol at -20°C/-80°C, avoiding repeated freeze-thaw cycles

For reconstitution, it is recommended to briefly centrifuge the vial before opening and reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol for long-term storage stability .

What experimental approaches can determine the membrane topology of SH1116?

Determining the exact topology of membrane proteins like SH1116 requires multiple complementary techniques:

• Computational prediction: Tools like TMHMM, Phobius, or TOPCONS can predict transmembrane regions based on the amino acid sequence

• Protease accessibility assays: Limited proteolysis followed by mass spectrometry to identify exposed regions

• Cysteine scanning mutagenesis: Systematic replacement of residues with cysteine followed by accessibility studies

• Fluorescence microscopy: GFP-fusion constructs at various positions to determine cytoplasmic vs. periplasmic localization

• Cryo-EM or X-ray crystallography: For high-resolution structural determination if the protein can be stabilized

By combining these approaches, researchers can develop a comprehensive model of how SH1116 is oriented within the membrane, which is crucial for understanding its potential interactions with other cellular components .

How can researchers effectively study protein-protein interactions involving SH1116?

Membrane proteins like SH1116 present unique challenges for interaction studies. A multi-method approach is recommended:

• Co-immunoprecipitation with crosslinking: Chemical crosslinkers can trap transient interactions before solubilization

• Bacterial two-hybrid systems: Modified for membrane protein analysis

• Surface plasmon resonance (SPR): For quantitative binding kinetics using purified components

• Proximity labeling approaches: BioID or APEX2 fusions to identify neighboring proteins in the native environment

Recent research on membrane protein assembly has identified complexes like the PAT (protein associated with the translocon) that assist in proper assembly of multi-spanning membrane proteins . Using chemical crosslinkers to trap factors involved in assembly has proven valuable in identifying previously uncharacterized interaction partners, which may also be applicable to studying SH1116 interactions .

How does SH1116 contribute to S. haemolyticus virulence and hospital adaptation?

While specific functions of SH1116 itself are still being elucidated, research on S. haemolyticus provides context for understanding potential roles of membrane proteins in virulence:

• Clinical S. haemolyticus isolates show distinct genetic signatures compared to commensal strains, including differences in membrane-associated proteins

• Hospital-adapted strains typically possess genes related to biofilm formation and antibiotic resistance

The comparison between clinical and commensal S. haemolyticus isolates reveals:

As a membrane protein, SH1116 may participate in adaptation to hospital environments, potentially contributing to surface properties that affect colonization or biofilm formation .

How do genomic variations in SH1116 differ between clinical and commensal isolates?

Comparative genomic analysis between clinical and commensal S. haemolyticus isolates reveals distinct phylogenetic clustering . While specific variations in SH1116 are not detailed in the search results, broader patterns in membrane proteins show:

• Clinical isolates often contain distinct conserved differences in surface-associated genes compared to commensal isolates

• Horizontal gene transfer appears to be a major driver in the evolution of clinical isolates

• Genetic rearrangements and beneficial point mutations in surface-associated genes are more common in hospital-adapted strains

For comprehensive analysis of SH1116 variations across strains, researchers should consider:

• Whole genome sequencing of diverse isolates

• Selection pressure analysis using dN/dS ratios

• Structural modeling to predict functional impacts of observed variations

• Experimental validation through site-directed mutagenesis

What are the challenges in determining the structure of SH1116 and strategies to overcome them?

Membrane proteins like SH1116 present significant structural biology challenges:

• Detergent selection: Screening multiple detergents or nanodiscs/amphipols for optimal stability

• Crystallization barriers: Use of antibody fragments or fusion partners to increase polar surface area

• Conformational heterogeneity: Ligand or inhibitor binding to stabilize a single conformation

• Alternative approaches: Cryo-EM or solid-state NMR when crystallization proves difficult

Researchers should consider implementing the lipidic cubic phase (LCP) method, which has proven successful for many membrane proteins. For intrinsically disordered regions, hydrogen/deuterium exchange mass spectrometry (HDX-MS) can provide valuable conformational information even without a crystal structure.

How can SH1116 be leveraged as a potential therapeutic target?

As S. haemolyticus emerges as a reservoir of antibiotic resistance , membrane proteins like SH1116 represent potential novel therapeutic targets. Strategic approaches include:

• High-throughput screening: Developing assays to identify small molecule binders

• Structure-based drug design: Once structural information is available

• Immunotherapeutic approaches: Evaluating accessibility of epitopes for antibody targeting

• Resistance mechanism analysis: Determining if SH1116 contributes directly or indirectly to resistance phenotypes

The increasing clinical relevance of S. haemolyticus as a nosocomial pathogen with multi-drug resistance emphasizes the importance of novel target exploration. Membrane proteins often serve essential functions that can be exploited for antimicrobial development.

How can researchers effectively study the role of SH1116 in the context of bacterial membrane organization?

Advanced imaging and biophysical techniques can elucidate SH1116's role in membrane organization:

• Super-resolution microscopy: Techniques like PALM/STORM to visualize protein clustering in bacterial membranes

• FRET analysis: To detect protein-protein interactions within the membrane

• Native mass spectrometry: For analyzing intact membrane protein complexes

• Neutron reflectometry: To determine protein orientation and insertion depth

Understanding membrane organization is critical as proper assembly of membrane proteins is essential for bacterial survival. Recent discoveries of chaperones that assist membrane protein assembly, such as the PAT complex, highlight the complex machinery involved in ensuring correct topology and folding .