Recombinant Rhodococcus erythropolis UPF0060 membrane protein RER_49640 (RER_49640)

What is the UPF0060 membrane protein RER_49640?

The UPF0060 membrane protein RER_49640 is a 108-amino acid membrane protein from Rhodococcus erythropolis. It belongs to the UPF0060 protein family, a group of uncharacterized proteins found across various bacterial species. The protein is encoded by the RER_49640 gene in R. erythropolis and contains a transmembrane domain characteristic of membrane-associated proteins . R. erythropolis is a Gram-positive actinomycete with high G+C content, capable of morphological differentiation in response to environmental conditions .

What expression systems are recommended for recombinant RER_49640 production?

• Expression should be conducted under tightly-controlled growth conditions

• Cells should be harvested prior to glucose exhaustion, just before the diauxic shift

• The most rapid growth conditions are not necessarily optimal for membrane protein production

• Expression levels may not correspond directly to mRNA levels, but rather relate to differential expression of genes involved in membrane protein secretion and cellular physiology

How should reconstituted RER_49640 be stored for maximum stability?

For optimal stability of recombinant RER_49640:

• Upon receipt, the lyophilized protein should be briefly centrifuged to bring contents to the bottom of the vial

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

• Add glycerol to a final concentration of 5-50% (default recommendation is 50%)

• Aliquot for long-term storage at -20°C/-80°C to avoid repeated freeze-thaw cycles

• For short-term use, working aliquots can be stored at 4°C for up to one week

What purification strategies yield highest purity for His-tagged RER_49640?

While specific optimization protocols for RER_49640 are not detailed in the provided sources, the following methodological approach is recommended based on best practices for His-tagged membrane proteins:

• Initial Extraction: Solubilize membrane fractions using appropriate detergents (typically mild non-ionic detergents like DDM or LDAO)

• IMAC Purification: Utilize Ni-NTA or similar metal affinity chromatography with an imidazole gradient

• Buffer Optimization: The protein is supplied in Tris/PBS-based buffer with 6% Trehalose at pH 8.0, suggesting this condition supports stability

• Quality Control: Purity assessment via SDS-PAGE should confirm >90% purity, as specified for commercially available recombinant RER_49640

How do different expression conditions affect RER_49640 yield and functionality?

Membrane protein production is highly sensitive to expression conditions. Research with recombinant membrane proteins indicates:

• Growth phase at harvest is critical: cells should be harvested just before the diauxic shift

• Tightly-controlled growth conditions in bioreactors provide more reliable yields compared to shake flasks

• Slower growth conditions often yield better protein production than maximum growth rate conditions

• Temperature, media composition, and inducer concentration all require systematic optimization

• Expression levels do not necessarily correlate with corresponding mRNA levels

A systematic approach using Design of Experiments (DoE) methodology is recommended to identify optimal conditions for RER_49640 expression.

What analytical methods can confirm proper folding and functionality of recombinant RER_49640?

To assess the structural integrity and functionality of recombinant RER_49640, researchers should consider a multi-method approach:

• Circular Dichroism (CD) Spectroscopy: To evaluate secondary structure content and confirm proper folding

• Size Exclusion Chromatography (SEC): To assess oligomeric state and homogeneity

• Thermal Stability Assays: Similar to those used for Rpf proteins from R. erythropolis, incubating the protein at different temperatures (20°C-80°C) to determine stability thresholds

• Metal Ion Effects: Testing the influence of various metal ions (such as Zn²⁺, Mg²⁺, Co²⁺, Ca²⁺, and Mn²⁺) at 0.1 mmol/L concentration on protein stability and activity

How does the biological context of R. erythropolis inform RER_49640 research?

Understanding the native biological context provides valuable insights for RER_49640 research:

• R. erythropolis strains such as PR4 were isolated from the Pacific Ocean at 1,000m depth and can degrade various alkanes and methylbenzenes

• These bacteria produce extracellular polysaccharides (EPSs) that contribute to tolerance against organic solvents

• Their genomes contain numerous secondary metabolism genes and EPS biosynthesis genes

• The metabolic versatility of R. erythropolis suggests membrane proteins like RER_49640 may be involved in substrate transport or environmental sensing

Experimental designs should consider these contextual factors, particularly when investigating potential functional roles.

What are the common pitfalls in membrane protein reconstitution and how can they be addressed?

Membrane protein reconstitution presents several challenges that researchers should anticipate:

• Protein Aggregation: Use freshly reconstituted protein and maintain appropriate detergent concentrations above their critical micelle concentration (CMC)

• Activity Loss: The recommended reconstitution protocol for RER_49640 involves using deionized sterile water and addition of glycerol (5-50%) for stability

• Reproducibility Issues: Standardize all reconstitution steps including centrifugation, buffer composition, and temperature

• Storage Problems: Repeated freeze-thaw cycles significantly reduce membrane protein activity; aliquot upon reconstitution and avoid multiple freeze-thaw cycles

How can researchers distinguish between effects of the His-tag and intrinsic properties of RER_49640?

To differentiate between tag artifacts and native protein properties:

• Comparative Studies: Express both tagged and tag-cleaved versions using proteolytic removal via engineered cleavage sites

• Control Experiments: Include appropriate controls with different tag positions (N-terminal vs. C-terminal)

• Functional Assays: Compare activity metrics between tagged and untagged versions where possible

• Structural Analysis: Assess whether the tag influences membrane insertion or protein folding through techniques like limited proteolysis or CD spectroscopy

What approaches enable successful integration of RER_49640 into model membrane systems?

For functional studies, proper integration into model membranes is critical:

• Liposome Reconstitution: Gradually remove detergent through dialysis or adsorption

• Nanodisc Formation: Incorporate protein into membrane scaffold protein (MSP)-bound lipid bilayers

• Supported Lipid Bilayers: Use for surface-sensitive techniques like atomic force microscopy

• Lipid Composition Optimization: Test various lipid compositions reflecting the native R. erythropolis membrane environment

How can the membrane topology of RER_49640 be experimentally determined?

Determining the precise membrane topology of RER_49640 requires multiple complementary approaches:

• Computational Prediction: Begin with hydropathy analysis and topology prediction algorithms

• Cysteine Scanning Mutagenesis: Introduce individual cysteine residues and assess accessibility

• Protease Protection Assays: Determine exposed regions through limited proteolysis

• Fluorescence Spectroscopy: Use environment-sensitive fluorophores to identify membrane-embedded regions

• Antibody Accessibility Studies: Generate antibodies against specific epitopes and test accessibility in intact vs. permeabilized systems

What structural homology does RER_49640 share with other characterized membrane proteins?

While specific structural information for RER_49640 is limited, a methodological approach to homology analysis would involve:

• Sequence-based comparisons with other UPF0060 family proteins

• Secondary structure prediction and comparison with characterized membrane proteins

• Identification of conserved motifs that might indicate functional regions

• Threading and homology modeling to generate structural hypotheses

• Experimental validation of predicted structural features

How might temperature and metal ions affect RER_49640 stability and function?

Based on studies with other proteins from R. erythropolis, particularly the Rpf proteins, researchers should consider:

• Temperature Stability: Test stability at various temperatures (20°C-80°C) for 30 minutes followed by activity measurements

• Metal Ion Effects: Evaluate the influence of metal ions including Zn²⁺, Mg²⁺, Co²⁺, Ca²⁺, and Mn²⁺ at 0.1 mmol/L concentration

• Thermal Denaturation Profiles: Generate thermal denaturation curves to identify transition temperatures

• Metal Binding Sites: Investigate potential metal binding sites through mutational analysis of conserved residues

How does research on RER_49640 contribute to understanding membrane protein expression systems?

Research on RER_49640 expression contributes to broader membrane protein production knowledge:

• Optimization strategies for RER_49640 may inform approaches for other challenging membrane proteins

• Understanding how growth conditions affect RER_49640 expression adds to the knowledge base for membrane protein production

• The relationship between harvest timing and protein quality reinforces the importance of physiological state in expression systems

• Systematic approaches to RER_49640 production illustrate the value of controlled bioreactor conditions over shake flask cultivations

What are the most promising directions for elucidating the biological function of RER_49640?

To determine the biological function of this uncharacterized protein:

• Gene Knockout Studies: Generate RER_49640 deletion mutants in R. erythropolis and assess phenotypic changes

• Transcriptomic Analysis: Identify conditions that alter RER_49640 expression

• Interactome Studies: Use pull-down assays to identify protein-protein interactions

• Comparative Genomics: Analyze conservation and genetic context across related species

• Environmental Response Studies: Evaluate expression changes under different growth conditions, particularly those mimicking the natural environment of R. erythropolis