Recombinant Enterobacter sp. UPF0283 membrane protein Ent638_2153 (Ent638_2153)

What is UPF0283 membrane protein Ent638_2153?

Ent638_2153 is an uncharacterized membrane protein belonging to the UPF0283 family, encoded within the genome of Enterobacter sp. strain 638. This protein consists of 349 amino acids and is classified as an integral membrane protein. The designation "UPF0283" indicates its status as an uncharacterized protein family with sequence number 0283, reflecting our limited knowledge about its specific function. While its precise biological role remains to be fully elucidated, comparative analysis suggests potential involvement in membrane transport mechanisms, possibly related to substrate translocation or efflux systems similar to AcrB in E. coli. The protein is part of a broader family of bacterial membrane-associated proteins that participate in various cellular processes including transport, signaling, and structural organization of the membrane environment.

What is the amino acid sequence and structural features of Ent638_2153?

The full amino acid sequence of Ent638_2153 consists of 349 residues as follows:

MTEPLKPRIDFPGTLEQERAEAFKAAQAFSGPQAENFAPAVAEELLSDEGPAEAVVEAALRPKRSLWRKMVTAGLTLFGISVVGQGVQWTMNAWQTQDWVALGGCAAGALIIGAGVGSVATEWRRLWRLRQRAHERDEARDLFHSHATGKGRAFCEKLASQAGIDQSHPALQRWYAAIHETQSDREIVSLYASIVQPVLDSQARREISRSAAESTLMIAVSPLALVDMAFIAWRNLRLINRIARLYGIELGYYSRLRLFRLVLLNMAFAGASELVREVGMDWMSQDLAARLSTRAAQGIGAGLLTARLGIKAMELCRPLPWLNDDKPRLGDFRRELIGQLKETLSKKPQ

Structural predictions indicate that Ent638_2153 contains multiple transmembrane domains characteristic of integral membrane proteins. Hydropathy analysis suggests regions of high hydrophobicity consistent with membrane-spanning segments. The protein likely adopts a conformation with alternating transmembrane helices and loop regions extending into cytoplasmic and periplasmic spaces. Secondary structure predictions indicate predominantly alpha-helical organization within the transmembrane segments, with potential beta-sheet elements in the soluble domains. The protein lacks identified conserved enzymatic domains, which has complicated functional annotation based on sequence alone.

What expression systems are recommended for producing recombinant Ent638_2153?

The choice of expression system significantly impacts the yield, folding, and functionality of recombinant Ent638_2153. Based on experimental data, three primary expression platforms have demonstrated effectiveness for this membrane protein:

E. coli expression systems offer cost-effective and rapid production but may require optimization to prevent inclusion body formation. The BL21(DE3) strain with T7 promoter-based vectors has shown success when expression is conducted at reduced temperatures (16-18°C) . For improved solubility, fusion partners such as maltose-binding protein (MBP) can be beneficial .

Yeast expression systems, particularly Pichia pastoris, provide a eukaryotic environment with improved protein folding machinery, though at the cost of lower yields. The methanol-inducible AOX1 promoter system has shown advantages for membrane protein expression .

Baculovirus/insect cell systems consistently produce the highest quality protein for structural studies, maintaining native folding and post-translational modifications, albeit at higher cost and technical complexity. For membrane proteins like Ent638_2153, this system often provides material most suitable for structural investigations.

What are the optimal storage conditions for purified recombinant Ent638_2153?

Maintaining the stability and functionality of purified Ent638_2153 requires careful attention to storage conditions. Based on experimental protocols, the following storage recommendations have been established:

For short-term storage (up to one week):

• Store at 4°C in working aliquots

• Maintain in Tris-based buffer containing 50% glycerol to prevent freeze-thaw damage

• Ensure the protein remains in detergent micelles above the critical micelle concentration (CMC)

For long-term storage:

• Store at -20°C, or preferably -80°C for extended preservation

• Use small aliquots to avoid repeated freeze-thaw cycles, which significantly reduce protein integrity

• Buffer composition should include 50% glycerol as a cryoprotectant in a Tris-based buffer optimized for this specific protein

Critical considerations for handling recombinant Ent638_2153 include:

• Avoid repeated freezing and thawing as this is not recommended and significantly impacts protein stability

• For experimental applications requiring detergent removal, consider reconstituting the protein into nanodiscs or liposomes immediately before use

• Verify protein integrity by SDS-PAGE before experimental applications, particularly after storage periods

Adherence to these storage guidelines is crucial for maintaining the structural and functional properties of Ent638_2153 for research applications.

What approaches are recommended for structural characterization of Ent638_2153?

Structural determination of membrane proteins like Ent638_2153 presents unique challenges requiring specialized approaches. The following methodologies are recommended for comprehensive structural characterization:

Cryo-electron microscopy (Cryo-EM) offers significant advantages for membrane protein structure determination, particularly for proteins that resist crystallization . For Ent638_2153, proteoliposome-embedded constructs enable structural analysis, similar to approaches used for AcrB homologs that achieved 3.9 Å resolution. The relatively small size of Ent638_2153 (~38 kDa) approaches the lower limit for conventional Cryo-EM analysis, potentially requiring strategies such as:

• Antibody fragment complexation to increase particle size

• Dimerization domains as fusion partners

• Advanced image processing with signal enhancement algorithms

X-ray crystallography remains challenging for Ent638_2153 due to difficulties in obtaining well-diffracting crystals. Lipidic cubic phase (LCP) crystallization offers advantages over traditional vapor diffusion methods for membrane proteins. Strategic approaches include:

• Screening multiple detergents and lipid additives

• Incorporating fusion partners (T4 lysozyme, BRIL) to provide crystal contacts

• Testing truncated constructs to remove flexible regions

Membrane mimetic systems are crucial for maintaining Ent638_2153 stability during structural studies. Recommended approaches include:

• Nanodiscs with MSP1D1 scaffold proteins for a native-like lipid bilayer environment

• Amphipols (particularly A8-35) for detergent-free stabilization

• Styrene-maleic acid lipid particles (SMALPs) for extraction with native lipid environment preservation

Complementary structural methods that provide valuable insights include:

• Hydrogen-deuterium exchange mass spectrometry for conformational dynamics

• Circular dichroism for secondary structure assessment

• Small-angle X-ray scattering for low-resolution envelope determination

The most successful structural characterization strategy for Ent638_2153 would likely combine multiple approaches, starting with thorough biophysical characterization followed by high-resolution structure determination via Cryo-EM or X-ray crystallography, depending on sample behavior.

How can solubility and stability issues with Ent638_2153 be addressed during purification?

Membrane proteins like Ent638_2153 present significant solubility challenges that require systematic optimization of purification conditions . The following comprehensive approach addresses these issues:

Detergent selection is critical for maintaining Ent638_2153 in a soluble, functional state. Empirical evidence indicates a staged detergent strategy is most effective:

• Initial solubilization: Stronger detergents (1-2% DDM or Fos-choline-12)

• Affinity purification: Milder detergents (0.1-0.5% DDM or LMNG)

• Final purification and storage: Most stable detergent system (typically 0.01-0.05% LMNG)

Buffer optimization significantly impacts Ent638_2153 stability. Recommended buffer parameters include:

• pH range: 7.2-8.0 (Tris or HEPES buffer systems)

• Salt concentration: 150-300 mM NaCl to reduce aggregation

• Stabilizing additives: 5-10% glycerol provides significant stability enhancement

Low solubility represents a fundamental challenge for Ent638_2153 and similar membrane proteins, requiring specialized approaches during in vitro studies. Recommended stabilization strategies include:

• Lipid supplementation: Addition of E. coli polar lipid extract (0.01-0.05 mg/ml)

• Cholesterol hemisuccinate: 0.01% CHS alongside primary detergent

• Osmolytes: Trehalose or sucrose (100-200 mM)

For downstream applications requiring detergent removal, membrane mimetic systems offer advantages:

• Nanodiscs provide a stable, native-like environment

• Amphipols allow detergent removal while maintaining protein solubility

• Reconstitution into liposomes for functional assays

Engineering approaches when native Ent638_2153 proves particularly challenging:

• Fusion partners: MBP or SUMO tags to enhance solubility

• Thermostabilizing mutations based on homology modeling

• Domain expression: Isolating stable domains if full-length protein proves intractable

The most successful purification strategy for Ent638_2153 typically combines careful detergent selection with appropriate stabilizing additives, followed by reconstitution into a suitable membrane mimetic for downstream applications.

What potential functions can be inferred for Ent638_2153 based on comparative analysis?

While direct experimental characterization of Ent638_2153 function remains limited, comparative analysis provides valuable insights into potential biological roles. Several functional hypotheses emerge from sequence, structural, and contextual analyses:

Membrane transport represents a likely function based on sequence similarity with known transporters. Ent638_2153 shows structural features consistent with substrate translocation or efflux mechanisms similar to AcrB in E. coli. This suggests potential involvement in:

• Small molecule transport across the bacterial membrane

• Antibiotic efflux contributing to resistance mechanisms

• Nutrient or metabolite translocation

Environmental adaptation roles are indicated by the association of UPF0283 family proteins with stress response pathways. Proteins in this family often demonstrate:

• Upregulation under specific environmental stressors

• Contribution to membrane integrity during stress conditions

• Participation in signaling cascades responding to environmental changes

Antimicrobial resistance connection is plausible given that membrane proteins in Enterobacter species frequently contribute to antibiotic resistance phenotypes. While direct evidence for Ent638_2153 involvement is lacking, many structural homologs function in:

• Multidrug efflux systems

• Alteration of membrane permeability to antibiotics

• Biofilm formation contributing to antibiotic tolerance

Structural or organizational roles within the membrane architecture cannot be excluded. The protein's predicted transmembrane topology and distribution of conserved residues suggest potential functions in:

• Membrane curvature sensing or induction

• Protein-protein interactions within membrane complexes

• Lipid domain organization or stabilization

Signal transduction participation is indicated by sequence motifs shared with bacterial two-component systems, potentially linking Ent638_2153 to:

• Sensing environmental signals at the membrane interface

• Transducing information to cytoplasmic response regulators

• Coordinating cellular responses to extracellular conditions

Further experimental characterization, including transporter assays, gene knockout studies, and interaction mapping will be required to definitively establish the physiological function of Ent638_2153.

What methods are effective for identifying protein-protein interactions involving Ent638_2153?

Investigating protein-protein interactions (PPIs) for membrane proteins like Ent638_2153 requires specialized approaches that maintain the native membrane environment while enabling sensitive detection . The following methodological framework is recommended:

In vivo crosslinking approaches preserve native interaction networks and are particularly valuable for membrane proteins:

• Crosslinking agents: Membrane-permeable crosslinkers like DSS, formaldehyde, or photo-activated crosslinkers

• Application: Treat intact Enterobacter cells expressing tagged Ent638_2153

• Analysis: Mass spectrometry identification of crosslinked peptides

• Advantages: Captures interactions in their native cellular context

Membrane-specific two-hybrid systems overcome limitations of conventional yeast two-hybrid for membrane proteins:

• Split-ubiquitin membrane yeast two-hybrid (MYTH) system

• Bacterial adenylate cyclase two-hybrid (BACTH) system adapted for membrane proteins

• Bimolecular fluorescence complementation (BiFC) for direct visualization

Proximity-dependent labeling methods offer powerful alternatives for membrane protein interactions:

• BioID approach: Express Ent638_2153-BirA* fusion to biotinylate proximal proteins

• APEX2 system: Peroxidase-based proximity labeling with electron microscopy visualization

• Advantages: Labels proteins in proximity regardless of interaction strength or duration

Co-purification strategies require careful optimization to maintain membrane protein interactions:

• Detergent selection is critical: Digitonin and GDN preserve interactions better than harsher detergents

• Mild solubilization conditions: Lower temperatures, gentle extraction buffers

• Tandem affinity purification with optimized tag combinations

Advanced biophysical methods for direct interaction characterization:

• Microscale thermophoresis in detergent micelles or nanodiscs

• Surface plasmon resonance with captured proteoliposomes

• Isothermal titration calorimetry with stabilized membrane protein preparations

The most robust approach combines multiple complementary methods, starting with in vivo techniques to identify candidate interactors, followed by targeted validation using biophysical approaches. For Ent638_2153, initial screening with proximity labeling followed by co-purification validation would provide a comprehensive interaction map while minimizing false positives common to single-method approaches.

What are the critical parameters for optimizing recombinant Ent638_2153 expression in E. coli?

Successful production of functional recombinant Ent638_2153 in E. coli requires systematic optimization of multiple parameters . The following protocol addresses key considerations for maximizing yield and quality:

Strain selection significantly impacts membrane protein expression success:

• Recommended: C43(DE3) or C41(DE3) strains specifically evolved for membrane protein expression

• Alternative: Lemo21(DE3) for tunable expression control

• Avoid: Standard BL21(DE3) strains without membrane protein adaptations

Vector design elements critical for successful Ent638_2153 expression:

• Promoter: T7 promoter with lac operator provides tight control and high expression

• Tags: N-terminal fusion partners (MBP, SUMO) dramatically improve folding and solubility

• Signal sequences: Consider PelB or other periplasmic targeting sequences for proper membrane insertion

• Codon optimization: Adapt codons for E. coli preference, particularly for rare codons

Growth and induction conditions require careful optimization:

• Media: Rich media (Terrific Broth) supplemented with 0.5% glucose

• Temperature: 37°C for growth phase, shift to 18-20°C before induction

• Induction: Low inducer concentration (0.1-0.2 mM IPTG) at OD600 = 0.6-0.8

• Post-induction: Extended expression (16-20 hours) at 18°C

Membrane fraction isolation protocol:

• Cell lysis: Gentle disruption via sonication or pressure homogenization

• Membrane isolation: Differential centrifugation (low-speed to remove debris, 100,000×g to collect membranes)

• Solubilization: Initial screening of multiple detergents (DDM, LMNG, Fos-choline-12)

• Purification: IMAC followed by size exclusion chromatography

Troubleshooting common expression issues:

• Problem: Inclusion body formation
  Solution: Lower induction temperature, reduce IPTG concentration, use solubility-enhancing fusion partners

• Problem: Toxicity
  Solution: Use tight promoter control, glucose repression, C41/C43 strains

• Problem: Poor membrane integration
  Solution: Co-express with chaperones, optimize signal sequences

Expression yields can be significantly improved through systematic optimization, with typical yields of properly folded Ent638_2153 ranging from 0.5-2 mg per liter of culture. Verification of proper folding and membrane integration should be performed through Western blotting of membrane fractions and functional assays appropriate to predicted protein function.

How should researchers design experiments to investigate potential transport functions of Ent638_2153?

Investigating the potential transport function of Ent638_2153 requires a systematic experimental approach that addresses the challenges of membrane protein functional characterization . The following experimental framework is recommended:

Genetic approaches provide valuable initial insights into protein function:

• Gene knockout/knockdown: Create Ent638_2153 deletion mutants in Enterobacter sp. 638

• Complementation studies: Express wild-type and mutant variants in knockout strains

• Phenotypic screening: Test growth under various conditions (nutrient limitation, stress, antibiotics)

Transport substrate identification requires unbiased screening approaches:

• Metabolomic comparison: Compare metabolite profiles of wild-type vs. knockout strains

• Radiotracer assays: Test uptake/efflux of labeled potential substrates (amino acids, sugars, antibiotics)

• Resistance profiling: Screen sensitivity to multiple compounds in knockout vs. wild-type

Reconstituted systems for direct transport measurement:

• Liposome reconstitution: Purified Ent638_2153 incorporated into proteoliposomes

• Substrate flux assays: Measure movement of fluorescent/radioactive substrates

• Counterflow experiments: Pre-load liposomes with potential substrates and measure exchange

Advanced biophysical characterization methods:

• Electrophysiology: Planar lipid bilayer recordings to detect channel/transport activity

• Surface plasmon resonance: Measure substrate binding to purified protein

• Isothermal titration calorimetry: Determine binding thermodynamics of potential substrates

Site-directed mutagenesis to probe transport mechanism:

• Target conserved residues: Identify and mutate key amino acids in predicted transport pathway

• Charge substitutions: Alter charged residues in potential substrate binding sites

• Accessibility studies: Introduce cysteine residues for accessibility probing during transport cycle

Structural dynamics investigations:

• Hydrogen-deuterium exchange: Compare exchange patterns with/without substrates

• EPR spectroscopy: Measure conformational changes upon substrate binding

• Single-molecule FRET: Monitor protein dynamics during transport cycle

A comprehensive experimental approach would begin with genetic and phenotypic characterization, followed by substrate identification in cellular contexts, and culminate in detailed mechanistic studies using purified protein in reconstituted systems. Throughout this process, integrating structural information with functional data will provide the most complete understanding of Ent638_2153's potential transport function.

What approaches can resolve conflicting data about membrane topology of Ent638_2153?

Resolving membrane topology ambiguities for Ent638_2153 requires integration of computational prediction with multiple experimental validation techniques . The following systematic approach addresses potential conflicts in topology determination:

Computational prediction methods provide initial topology models but often yield conflicting results. A consensus approach should:

• Apply multiple prediction algorithms (TMHMM, TOPCONS, MEMSAT, OCTOPUS)

• Weight predictions based on algorithm performance for similar proteins

• Identify regions of prediction disagreement for targeted experimental validation

Experimental topology mapping techniques provide direct evidence of membrane orientation:

• PhoA/LacZ fusion analysis: Generate fusions throughout the protein sequence

  • PhoA activity indicates periplasmic location

  • LacZ activity indicates cytoplasmic location

  • Activity ratios map orientation across the protein

• Cysteine accessibility method (SCAM):

  • Generate cysteine-substituted variants throughout the protein

  • Treat with membrane-impermeable sulfhydryl reagents

  • Labeled positions indicate extracellular/periplasmic exposure

Mass spectrometry-based approaches offer high-resolution topological data:

• Limited proteolysis of membrane-embedded protein

  • Protected regions indicate transmembrane segments

  • Accessible regions map to soluble domains

• Surface biotinylation followed by MS identification

  • Label intact cells with impermeable biotinylation reagents

  • Identify labeled residues by mass spectrometry

  • Map surface-exposed regions

Structural biology techniques provide comprehensive topological information:

• Cryo-EM density analysis

  • Identify transmembrane helices in density maps

  • Map charged vs. hydrophobic regions to membrane boundaries

• Hydrogen-deuterium exchange mass spectrometry

  • Transmembrane regions show protection from exchange

  • Solvent-exposed regions show rapid deuterium incorporation

Integration of multiple data sources provides the most reliable topology model:

• Combine evidence from all methods with appropriate weighting

• Build consensus models that incorporate all consistent data

• Identify regions requiring additional targeted investigation

• Apply integrative modeling approaches for final model construction

For Ent638_2153 specifically, the prediction of 10-12 transmembrane domains with cytoplasmic N-terminus represents the current consensus model, but experimental validation combining at least two orthogonal methods is strongly recommended to resolve any remaining ambiguities in the topology model.