Recombinant Xanthobacter autotrophicus UPF0060 membrane protein Xaut_1380 (Xaut_1380)

What is Xanthobacter autotrophicus UPF0060 membrane protein Xaut_1380?

Xanthobacter autotrophicus UPF0060 membrane protein Xaut_1380 is a full-length membrane protein (106 amino acids) from the bacterium Xanthobacter autotrophicus. It belongs to the UPF0060 protein family, a group of uncharacterized membrane proteins found in various bacterial species. The protein is encoded by the Xaut_1380 gene in Xanthobacter autotrophicus and has a UniProt ID of A7IF35 . As a membrane protein, it contains hydrophobic regions that allow it to integrate into the bacterial cell membrane, though its precise physiological function remains under investigation. The classification as a UPF (Uncharacterized Protein Family) indicates that while the protein has been identified through genomic sequencing, its biological role has not been fully characterized through experimental studies.

What expression systems are suitable for recombinant production of Xaut_1380?

For E. coli expression, consider using strains specifically designed for membrane protein expression (C41, C43) and expression vectors with tightly controlled promoters to minimize potential toxicity effects. Fusion partners such as MBP (Maltose Binding Protein) or SUMO may also improve solubility and expression levels for challenging membrane proteins .

How should Xaut_1380 be stored and handled for optimal stability?

Based on established protocols for this specific protein, the following storage and handling recommendations should be followed :

• Short-term storage (up to one week): Store working aliquots at 4°C

• Long-term storage: Store at -20°C or preferably -80°C

• Buffer conditions: Tris/PBS-based buffer, pH 8.0, containing 6% trehalose

• Glycerol addition: Addition of 5-50% glycerol (with 50% being optimal) for cryoprotection

• Aliquoting: Divide into single-use aliquots to avoid repeated freeze-thaw cycles

• Reconstitution: Briefly centrifuge vials before opening to bring contents to the bottom

For optimal stability, it is critical to avoid repeated freeze-thaw cycles as they can lead to protein denaturation and aggregation, particularly for membrane proteins which are inherently less stable than soluble proteins when removed from their native lipid environment .

What is the recommended reconstitution protocol for lyophilized Xaut_1380?

The recommended reconstitution protocol for lyophilized Xaut_1380 involves the following steps :

• Briefly centrifuge the vial containing lyophilized protein to ensure all material is 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 5-50% (50% is recommended) for stability

• Aliquot the reconstituted protein into single-use volumes

• Store reconstituted aliquots at -20°C/-80°C for long-term storage

For membrane proteins like Xaut_1380, consider the following additional steps to maintain functionality:

• Addition of mild detergents (e.g., 0.05% DDM or 0.1% CHAPS) to prevent aggregation

• Inclusion of reducing agents (e.g., 1mM DTT) if the protein contains cysteine residues

• Use of sonication or gentle vortexing rather than vigorous mixing to avoid protein denaturation

What are the challenges in expressing full-length Xaut_1380 in prokaryotic systems?

Expression of full-length membrane proteins like Xaut_1380 in prokaryotic systems presents several challenges that require careful experimental design to overcome:

• Hydrophobicity and membrane integration: The hydrophobic nature of membrane proteins can lead to toxicity in expression hosts, as overexpression can disrupt membrane integrity. Analysis of the Xaut_1380 sequence reveals highly hydrophobic regions typical of membrane proteins .

• Codon usage bias: Differences in codon preference between Xanthobacter autotrophicus and expression hosts like E. coli can lead to translational pausing and reduced expression. This may be particularly problematic for rare codons occurring in clusters .

• Protein folding and stability: Membrane proteins often require specific chaperones or membrane environments for proper folding. In heterologous expression systems, improper folding can lead to aggregation or degradation.

• Toxicity to expression host: Overexpression of membrane proteins can be toxic to the host cell, necessitating tightly controlled expression systems.

• Translation initiation problems: For Xaut_1380, potential problems with truncated products may occur due to internal translation initiation sites or proteolysis. The use of dual fusion tags (N and C-terminal) can help identify full-length protein products .

Methodological approaches to address these challenges include:

• Use of specialized E. coli strains (C41/C43) designed for membrane protein expression

• Reduced induction temperature (16-20°C) to slow protein synthesis and improve folding

• Codon optimization of the Xaut_1380 gene for the expression host

• Addition of specific lipids or membrane-mimicking detergents to the growth media

How can protein-protein interactions of Xaut_1380 be investigated?

Investigating protein-protein interactions for membrane proteins like Xaut_1380 requires specialized approaches that account for their hydrophobic nature:

For Xaut_1380 specifically, its small size (106 amino acids) and membrane localization suggest approaches that minimize disruption of its native environment. The His-tagged version of the protein provides a convenient handle for pull-down experiments, while incorporation into nanodiscs or liposomes can maintain a membrane-like environment for interaction studies .

What experimental approaches are recommended for studying the membrane topology of Xaut_1380?

Understanding the membrane topology of Xaut_1380 is crucial for elucidating its function. Several complementary experimental approaches can be employed:

• Computational prediction: Initial topology models can be generated using algorithms that predict transmembrane regions based on hydrophobicity analysis of the amino acid sequence (MTLPAFLFAALGEIAGCFAVWHVVRLGGSHWWLLPGIVSLAAFAYALTFVEAEAAGRAFA AYGGIYILSSLVWMWTVEGVRPDRWDATGAALCLAGAAVIVFGPRG) .

• Cysteine scanning mutagenesis: Systematic replacement of residues with cysteine followed by accessibility studies using membrane-permeable and impermeable thiol-reactive reagents.

• Fluorescence protease protection (FPP) assay: Tagging different regions of Xaut_1380 with fluorescent proteins and determining their susceptibility to protease digestion from either side of the membrane.

• Antibody accessibility studies: Generation of antibodies against specific epitopes of Xaut_1380 and testing their ability to bind to intact cells versus permeabilized cells.

• Reporter fusion analysis: Creation of fusion proteins with topology-indicating reporters (such as PhoA, GFP, or LacZ) at different positions in the Xaut_1380 sequence.

The small size of Xaut_1380 (106 amino acids) suggests it may have 2-4 transmembrane domains. A combined approach using both computational predictions and experimental validation would provide the most reliable topology model.

How can contradictions in experimental results regarding Xaut_1380 be resolved?

When faced with contradictory experimental results concerning Xaut_1380, a systematic approach to resolution includes:

• Methodological analysis: Carefully examine differences in experimental conditions, protein preparation methods, and assay systems that might explain divergent results .

• Protein quality assessment: Verify protein integrity through techniques such as:

  • SDS-PAGE to confirm molecular weight and purity

  • Mass spectrometry to verify sequence integrity

  • Circular dichroism to assess secondary structure

  • Size exclusion chromatography to detect aggregation

• Controlled variable experiments: Design experiments that systematically test one variable at a time while keeping all others constant to isolate the source of contradictions.

• Independent validation: Use complementary techniques to verify results from multiple methodological angles.

• Statistical rigor: Apply appropriate statistical tests to determine if apparent contradictions are statistically significant or within the range of experimental variation.

• Reproducibility assessment: Implement standardized protocols across different labs or researchers to confirm reproducibility.

A common source of contradictions in membrane protein research is the detergent or lipid environment used, which can dramatically impact protein conformation and function. For Xaut_1380, standardizing the buffer composition (Tris/PBS-based, pH 8.0) and detergent conditions across experiments may help resolve contradictions.

What are the considerations for designing site-directed mutagenesis experiments for Xaut_1380?

Site-directed mutagenesis is a powerful approach to probe structure-function relationships in Xaut_1380. Key considerations include:

• Target selection:

  • Conserved residues across UPF0060 family members

  • Charged residues within or adjacent to predicted transmembrane domains

  • Potential functional motifs

  • Residues in predicted loops connecting transmembrane segments

• Mutation design strategy:

  • Conservative substitutions (e.g., Leu→Ile) to test structural roles

  • Non-conservative substitutions (e.g., Leu→Asp) to test functional roles

  • Alanine scanning of specific regions to identify essential residues

  • Cysteine substitutions for accessibility and crosslinking studies

• Control mutations:

  • Include mutations in non-conserved, non-functional regions as negative controls

  • Design mutations that preserve hydrophobicity profiles for transmembrane regions

• Expression and analysis considerations:

  • Verify that mutations don't disrupt protein expression or trafficking

  • Ensure proper folding of mutant proteins

  • Develop functional assays specific to hypothesized role of Xaut_1380

For Xaut_1380, particular attention should be paid to the regions between amino acids 20-40 and 70-90, which contain sequences consistent with transmembrane domains, as well as the more hydrophilic regions that may be involved in protein-protein interactions or substrate binding.