Recombinant UPF0233 membrane protein CE0031 (CE0031)

What is Recombinant UPF0233 membrane protein CE0031 and what is its function?

Recombinant UPF0233 membrane protein CE0031 (UniProt ID: Q8FUI7) is a small membrane protein from Corynebacterium efficiens that spans 90 amino acids. It's also known as Cell division protein CrgA (gene name: crgA) . The protein is typically produced with an N-terminal His tag to facilitate purification when expressed in E. coli systems. While the "UPF" designation indicates it belongs to an uncharacterized protein family, research suggests its involvement in cell division processes in Corynebacterium species. The protein likely functions within the membrane environment to facilitate cellular division, though specific molecular mechanisms remain under investigation.

What expression systems are recommended for UPF0233 membrane protein CE0031 production?

For optimal expression:

• Use the BL21ΔABCF strain, particularly for small outer membrane β-barrel proteins

• Grow cultures at 30°C rather than 37°C (the quadruple mutant grows better at lower temperatures)

• Use appropriate induction parameters based on your expression vector system

• Include proper tags (His-tag is common) to facilitate downstream purification

Note that growth behavior will differ from standard BL21(DE3) strains, with slower growth rates but potentially higher final protein yields for membrane proteins .

How should UPF0233 membrane protein CE0031 be reconstituted for experimental use?

Proper reconstitution of UPF0233 membrane protein CE0031 is critical for maintaining structural integrity and function. Follow this methodological approach:

• Centrifuge the lyophilized protein vial briefly to collect all material at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 50% (range of 5-50% is acceptable) to prevent freeze-thaw damage

• Aliquot into small volumes to minimize freeze-thaw cycles

• Store reconstituted protein at -20°C/-80°C for long-term storage, or at 4°C for up to one week for active experiments

The addition of glycerol is particularly important as membrane proteins are susceptible to denaturation during freeze-thaw cycles. For experiments requiring the native conformation, consider reconstitution into appropriate lipid bilayers or detergent micelles that mimic the membrane environment.

What are the optimal storage conditions for UPF0233 membrane protein CE0031?

UPF0233 membrane protein CE0031 requires specific storage conditions to maintain stability and activity:

Repeated freeze-thaw cycles significantly reduce protein quality and should be strictly avoided . For optimal results, immediately aliquot reconstituted protein into single-use volumes. The storage buffer composition (Tris/PBS-based buffer with 6% Trehalose, pH 8.0) helps maintain protein stability, but additional stabilizers like glycerol are required for freeze storage.

What experimental design approaches are recommended for studying UPF0233 membrane protein function?

When investigating UPF0233 membrane protein function, consider implementing these research design approaches:

• Comparative expression analysis: Express the protein in both standard BL21(DE3) and optimized BL21ΔABCF strains to assess expression efficiency and protein quality via Western blotting and activity assays

• Structured time-series experimentation: Implement interrupted time series (ITS) designs to evaluate protein function under varying conditions, measuring outcomes at multiple timepoints before and after interventions

• Stepped-wedge design: When testing multiple conditions or mutations, use a staggered implementation approach where all experimental units eventually receive the intervention but in a randomized sequence

• Membrane environment studies: Compare protein behavior in different membrane mimetics (detergent micelles, nanodiscs, liposomes) to identify optimal conditions for functional studies

• Biophysical measurements: Utilize the unique attributes of BL21ΔABCF strains that facilitate labeling experiments in the native membrane environment for direct biophysical measurements

For rigorous implementation science approaches, combine these methods with appropriate controls and replication to ensure reproducibility and statistical power .

How can researchers optimize the purification of UPF0233 membrane protein CE0031?

Purification of UPF0233 membrane protein CE0031 requires specialized approaches due to its membrane-embedded nature. Researchers should follow this advanced purification workflow:

• Expression optimization:

  • Use BL21ΔABCF strain for significantly improved expression

  • Implement a controlled induction protocol (temperature, inducer concentration, duration)

  • Monitor expression via Western blot using anti-His antibodies

• Cell lysis and membrane isolation:

  • Use gentle lysis methods (osmotic shock or enzymatic methods)

  • Separate membrane fractions via ultracentrifugation (100,000×g for 1 hour)

  • Wash membrane pellets to remove peripheral proteins

• Detergent solubilization:

  • Screen multiple detergents (e.g., DDM, LDAO, OG) for optimal solubilization

  • Maintain solubilization temperature at 4°C

  • Use gentle rotation rather than harsh agitation

• Affinity chromatography:

  • Utilize the N-terminal His-tag with Ni-NTA resin

  • Include detergent in all buffers at concentrations above CMC

  • Implement step gradients for washing and elution

• Quality assessment:

  • Verify purity via SDS-PAGE (>90% purity expected)

  • Confirm identity via Western blotting or mass spectrometry

  • Assess structural integrity via circular dichroism

This methodological approach accounts for the challenges specific to membrane proteins and leverages the His-tag design of the recombinant construct .

What are the challenges in structural characterization of UPF0233 membrane protein CE0031?

Structural characterization of UPF0233 membrane protein CE0031 presents several methodological challenges that researchers must address systematically:

• Protein stability considerations:

  • The small size (90 amino acids) makes the protein susceptible to aggregation

  • Transmembrane regions can destabilize in inappropriate detergent environments

  • The high hydrophobicity (evident from the sequence WYKVIMFAFMLVGLLWLVANYLVGPQ) requires specialized handling

• Crystallization barriers:

  • Membrane proteins typically have lower crystallization success rates

  • Detergent micelles can interfere with crystal contacts

  • The small size may provide insufficient crystal contacts

• NMR spectroscopy approaches:

  • Isotopic labeling requires optimized minimal media conditions in the expression system

  • Detergent selection critically impacts spectral quality

  • The multiple transmembrane regions can lead to signal overlap

• Cryo-EM considerations:

  • The small size (90 residues, ~10 kDa) falls below typical size limits for single-particle cryo-EM

  • Strategies like antibody labeling or fusion partners may be necessary

  • Specialized grid preparation techniques for membrane proteins are required

To overcome these challenges, researchers should consider:

• Using the optimized BL21ΔABCF strain for improved expression quality

• Applying lipid cubic phase or bicelle crystallization methods

• Implementing advanced labeling strategies for NMR spectroscopy

• Exploring newer detergent-free systems like styrene maleic acid lipid particles (SMALPs)

How can site-directed mutagenesis be applied to study UPF0233 membrane protein CE0031 function?

Site-directed mutagenesis represents a powerful approach for investigating UPF0233 membrane protein CE0031 function. Implement the following methodological workflow:

• Target identification:

  • Analyze the amino acid sequence (MPKAKVTKNSIAPVSSNPSANRTPVKINSTGTPMWYKVIMFAFMLVGLLWLVANYLVGPQIPFMNELDAWNYGIGFGLLIIGLLMTMGWR) for conserved residues

  • Identify transmembrane regions using prediction tools

  • Focus on charged residues within or adjacent to transmembrane domains

• Mutagenesis strategy:

  • Employ PCR-based methods similar to those used for OmpX modifications

  • Consider the Byrappa method for site-directed mutagenesis as demonstrated for OmpX-HA tag insertion

  • Design primers with 15-20 bp flanking sequences around the mutation site

• Expression comparison:

  • Express wild-type and mutant proteins in parallel

  • Use the BL21ΔABCF strain for optimal expression

  • Quantify expression levels using Western blotting with anti-His antibodies

• Functional assessment:

  • Develop membrane localization assays

  • Assess oligomerization state changes via crosslinking

  • Measure effects on cell division if expressing in Corynebacterium model systems

• Structural impact analysis:

  • Use circular dichroism to assess secondary structure perturbations

  • Apply limited proteolysis to identify structural changes

  • Consider thermal stability assays to quantify folding impacts

This systematic approach enables researchers to correlate specific amino acid positions with functional outcomes, thereby elucidating structure-function relationships for this membrane protein.

What common challenges arise during UPF0233 membrane protein CE0031 expression and how can they be resolved?

Researchers frequently encounter several challenges when expressing UPF0233 membrane protein CE0031. Here are methodological solutions to these common issues:

• Low expression levels:

  • Replace standard BL21(DE3) with BL21ΔABCF strain, which has shown significantly improved expression for membrane proteins

  • Lower induction temperature to 30°C instead of 37°C to match optimal growth conditions for ΔABCF strains

  • Extend induction time to compensate for slower growth of ΔABCF strains

• Protein aggregation:

  • Monitor salt concentration closely as ΔABCF strains showed differences in aggregation behavior in high-salt conditions

  • Add mild detergents during cell lysis to prevent aggregation of hydrophobic regions

  • Include 6% trehalose in buffers as used in the storage buffer formulation

• Poor solubilization:

  • Screen multiple detergent types and concentrations

  • Implement step-wise solubilization protocols starting with milder detergents

  • Consider native nanodiscs or SMALPs for extraction without conventional detergents

• Degradation during purification:

  • Add protease inhibitor cocktails throughout all purification steps

  • Maintain samples at 4°C throughout processing

  • Minimize time between cell harvest and protein purification

• Low protein purity:

  • Implement multi-step purification including ion exchange after initial His-tag purification

  • Consider size exclusion chromatography as a final polishing step

  • Validate using SDS-PAGE to achieve >90% purity as specified

These solutions address the specific challenges associated with this membrane protein while leveraging the advantages of optimized expression systems .

How can researchers verify the proper folding and activity of UPF0233 membrane protein CE0031?

Verifying proper folding and activity of UPF0233 membrane protein CE0031 requires multiple complementary approaches:

• Biochemical characterization:

  • Size exclusion chromatography to confirm monomeric state or defined oligomers

  • Circular dichroism spectroscopy to assess secondary structure content expected of membrane proteins

  • Thermal stability assays to determine melting temperature and stability parameters

• Membrane integration assessment:

  • Membrane fractionation followed by Western blotting to confirm localization

  • Protease protection assays to verify topology within membranes

  • Fluorescent labeling and microscopy to visualize membrane localization

• Functional verification:

  • For CrgA/CE0031's putative role in cell division, assess complementation in knockout models

  • Analyze protein-protein interactions with known cell division proteins

  • Measure effects on cell morphology when overexpressed

• Structural integrity checks:

  • Limited proteolysis to verify compact folding (properly folded proteins show resistance to digestion)

  • Tryptophan fluorescence to assess tertiary structure environment

  • Antibody recognition of conformational epitopes

• Activity assays:

  • As this is an UPF (uncharacterized protein family), design activity assays based on bioinformatic predictions

  • Consider lipid binding assays to assess membrane interaction properties

  • Measure oligomerization dynamics using FRET-based approaches

These methodological approaches provide a comprehensive assessment of protein quality beyond simple expression and purification verification .

What analytical methods are recommended for studying the membrane integration of UPF0233 CE0031?

Studying membrane integration of UPF0233 CE0031 requires specialized analytical methods that provide insights into protein-membrane interactions:

• Biophysical membrane association analyses:

  • Fluorescence spectroscopy of tryptophan residues (present in the sequence) to monitor membrane insertion

  • Differential scanning calorimetry to measure thermodynamic parameters of protein-membrane interactions

  • Surface plasmon resonance with immobilized lipid bilayers to quantify binding kinetics

• Structural membrane topology determination:

  • Cysteine scanning mutagenesis combined with accessibility assays

  • Hydrogen/deuterium exchange mass spectrometry to identify membrane-protected regions

  • Electron paramagnetic resonance spectroscopy with site-directed spin labeling

• Advanced microscopy techniques:

  • Super-resolution microscopy of fluorescently labeled protein to visualize membrane distribution

  • Atomic force microscopy of reconstituted proteoliposomes to visualize topography

  • Cryo-electron microscopy of membrane-embedded protein

• Computational approaches:

  • Molecular dynamics simulations to predict stable membrane orientations

  • Analysis of hydrophobic regions in the sequence WYKVIMFAFMLVGLLWLVANYLVGPQ that likely form transmembrane helices

  • Comparison with structurally characterized homologs to predict membrane interfaces

• Biochemical membrane integration assays:

  • Alkaline extraction to differentiate peripheral from integral membrane proteins

  • Protease protection assays to map exposed versus membrane-embedded domains

  • Crosslinking studies to identify adjacent membrane proteins in native environments

These methodological approaches provide complementary data about how CE0031 integrates into and functions within biological membranes, leveraging both conventional techniques and advanced technologies .

What future research directions are promising for UPF0233 membrane protein CE0031?

Several promising research directions could advance our understanding of UPF0233 membrane protein CE0031:

• Structural biology advancements:

  • Cryo-EM studies using new technologies optimized for small membrane proteins

  • X-ray crystallography utilizing the improved expression in BL21ΔABCF strains

  • Integrative structural biology combining multiple methods to resolve the complete structure

• Functional characterization:

  • Systematic interactome mapping in Corynebacterium to identify binding partners

  • Gene knockout/complementation studies to definitively establish physiological roles

  • Evolution of CrgA/CE0031 across bacterial species to understand conserved functions

• Methodological innovations:

  • Development of cell-free expression systems optimized for this specific membrane protein

  • Application of non-canonical amino acid incorporation for specialized biophysical studies

  • Machine learning approaches to predict functional sites based on sequence conservation

• Therapeutic potential exploration:

  • Assessment of CE0031 as a potential antibiotic target in pathogenic Corynebacterium species

  • Development of specific inhibitors if essential functions are confirmed

  • Exploration of immunological recognition in host-pathogen interactions

• Biotechnological applications:

  • Engineering CE0031 as a potential membrane-targeting module for synthetic biology

  • Development of CE0031-based biosensors for detecting membrane perturbations

  • Exploration as a model system for membrane protein folding studies