Recombinant Cellvibrio japonicus UPF0060 membrane protein CJA_3703 (CJA_3703)

What is Cellvibrio japonicus UPF0060 membrane protein CJA_3703?

Cellvibrio japonicus UPF0060 membrane protein CJA_3703 is a membrane-associated protein encoded by the CJA_3703 gene in the genome of Cellvibrio japonicus, a saprophytic soil bacterium known for its ability to degrade plant cell wall polysaccharides. The protein belongs to the UPF0060 family of membrane proteins, a group of proteins with conserved structure but largely uncharacterized function. The full-length protein consists of 108 amino acids and has a UniProt ID of B3PHW0 . C. japonicus was originally isolated from Japanese soil and has gained research interest due to its extensive repertoire of enzymes for plant cell wall degradation .

When working with this protein, researchers should note that it is typically available as a recombinant protein with various tags for purification and detection purposes. The most common form is His-tagged recombinant CJA_3703 expressed in E. coli expression systems .

How is recombinant CJA_3703 typically expressed and purified?

Recombinant CJA_3703 protein is most commonly expressed in E. coli expression systems with an N-terminal His-tag to facilitate purification . The expression in E. coli offers several advantages, including high yield, cost-effectiveness, and well-established protocols. The His-tag enables efficient purification using immobilized metal affinity chromatography (IMAC).

For optimal expression and purification:

• Clone the CJA_3703 gene into an appropriate expression vector containing an N-terminal His-tag sequence.

• Transform the construct into an E. coli expression strain suitable for membrane proteins (e.g., C41(DE3) or C43(DE3)).

• Induce protein expression under optimized conditions (typically IPTG induction at OD600 of 0.6-0.8).

• Harvest cells and disrupt them using sonication or French press.

• Solubilize membrane fractions using appropriate detergents.

• Purify using Ni-NTA affinity chromatography.

• Perform size exclusion chromatography for further purification if needed.

• Verify purity using SDS-PAGE (>90% purity is typically achieved) .

Alternative expression systems such as yeast, baculovirus, and mammalian cells are also available for producing recombinant CJA_3703, especially when specific post-translational modifications or folding requirements are needed .

What are the optimal storage and handling conditions for recombinant CJA_3703?

For optimal storage and handling of recombinant CJA_3703 protein, follow these research-backed protocols:

The protein is typically supplied as a lyophilized powder and should be stored at -20°C/-80°C upon receipt . Before opening, briefly centrifuge the vial to bring the contents to the bottom. For reconstitution, it is recommended to use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL .

For long-term storage, add 5-50% glycerol (with 50% being the default recommendation) and aliquot the protein solution to avoid repeated freeze-thaw cycles, which can significantly damage protein structure and function . Working aliquots can be stored at 4°C for up to one week .

The storage buffer typically consists of Tris/PBS-based buffer with 6% trehalose at pH 8.0 . Trehalose serves as a stabilizing agent that helps maintain protein integrity during freeze-thaw cycles and storage.

When designing experiments with CJA_3703, consider the following stability considerations:

• Avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C

• Use appropriate buffer conditions that mimic the protein's native environment

• When performing assays, consider the membrane-associated nature of the protein and include appropriate detergents if necessary

What experimental approaches are recommended for studying membrane protein function in CJA_3703?

Studying the function of membrane proteins like CJA_3703 requires specialized approaches that address their hydrophobic nature and structural complexity. Here are methodological recommendations for functional studies:

• Liposome Reconstitution: Reconstitute purified CJA_3703 into liposomes composed of lipids that mimic the bacterial membrane composition. This approach allows for functional studies in a membrane-like environment.

• Site-Directed Mutagenesis: Identify conserved residues in the CJA_3703 sequence and create point mutations to assess their importance for function. This can be particularly useful for studying the transmembrane domains and any potential active sites.

• Protein-Protein Interaction Studies: Use techniques such as pull-down assays, co-immunoprecipitation, or crosslinking followed by mass spectrometry to identify potential interaction partners of CJA_3703 in the context of C. japonicus biology.

• Localization Studies: Develop fluorescently tagged versions of CJA_3703 to study its subcellular localization and potential dynamic behavior in live cells.

• Structural Studies: Consider using methods such as cryo-electron microscopy or solution NMR with detergent-solubilized protein to determine the three-dimensional structure.

When designing these experiments, it's important to consider the unique challenges posed by membrane proteins, including their hydrophobicity, potential for aggregation, and sensitivity to detergents.

How does CJA_3703 potentially relate to the plant cell wall degradation capabilities of C. japonicus?

Cellvibrio japonicus is renowned for its extensive repertoire of enzymes involved in plant cell wall degradation. The genome of C. japonicus encodes approximately 130 predicted glycoside hydrolases that target major structural and storage plant polysaccharides . In this context, understanding the potential role of CJA_3703 membrane protein requires careful experimental approaches.

While direct evidence linking CJA_3703 to plant cell wall degradation is not explicitly provided in the search results, several hypotheses can be explored:

• Transport Function: As a membrane protein, CJA_3703 might function in the transport of released sugars or oligosaccharides across the cell membrane during plant cell wall degradation.

• Sensing and Signaling: It may be involved in detecting plant cell wall components in the environment and triggering appropriate enzymatic responses.

• Enzyme Anchoring: CJA_3703 could serve as an anchor for secreted or cell-surface enzymes involved in polysaccharide degradation.

To investigate these possibilities, researchers should consider knockout or knockdown studies of the CJA_3703 gene, followed by phenotypic assays measuring growth on different plant polysaccharides. Comparative genomics with other plant biomass-degrading bacteria, such as Saccharophagus degradans (which shares approximately 50% of its plant-degradative apparatus with C. japonicus) , may also provide insights into conserved membrane proteins involved in this process.

What experimental designs are suitable for studying the role of CJA_3703 in bacterial adaptation to different carbon sources?

Investigating the role of CJA_3703 in bacterial adaptation to different carbon sources requires sophisticated experimental designs that integrate molecular genetics, biochemistry, and systems biology approaches. Here is a methodological framework:

• Experimental Design for Gene Expression Studies:

  • Implement a split-plot design where the main plot factor is the carbon source (e.g., glucose, xylose, arabinose, cellulose) and the subplot factor is the time point of sampling.

  • Use quantitative RT-PCR or RNA-Seq to monitor CJA_3703 expression levels under each condition.

  • Apply response surface methodology to optimize growth conditions and identify interaction effects between carbon source type and concentration .

• Knockout/Complementation Studies:

  • Create a CJA_3703 deletion mutant using homologous recombination or CRISPR-Cas9 technology.

  • Design a factorial experiment examining growth rates, lag phases, and maximum cell densities across multiple carbon sources.

  • Complement the mutation with wild-type and mutated versions of CJA_3703 to establish structure-function relationships.

• Proteomics Approach:

  • Use an incomplete block design to study the membrane proteome changes in wild-type versus CJA_3703 mutant strains under different carbon sources .

  • Apply stable isotope labeling techniques such as SILAC to quantitatively compare protein abundance.

  • Implement hierarchical clustering analysis to identify proteins with co-expression patterns similar to CJA_3703.

• Metabolic Flux Analysis:

  • Design experiments using 13C-labeled carbon sources to trace metabolic fluxes.

  • Compare flux distributions between wild-type and CJA_3703 mutant strains to identify metabolic pathways affected by the mutation.

These experimental designs should be analyzed using appropriate statistical methods, including ANOVA for factorial designs and response surface methodology for optimization experiments, as mentioned in the experimental design course materials .

How can structural and functional aspects of CJA_3703 be compared with similar membrane proteins in plant biomass-degrading bacteria?

Conducting a comprehensive comparative analysis of CJA_3703 with similar membrane proteins in other plant biomass-degrading bacteria requires an integrated approach combining bioinformatics, structural biology, and functional genomics:

• Sequence-Based Comparative Analysis:

  • Perform multiple sequence alignment of CJA_3703 with UPF0060 family proteins from other bacteria, particularly focusing on Saccharophagus degradans, which shares approximately 50% of its plant-degradative apparatus with C. japonicus .

  • Identify conserved residues and motifs that may indicate functional importance.

  • Construct phylogenetic trees to understand evolutionary relationships among these proteins.

• Structural Homology Modeling and Comparison:

  • Using the amino acid sequence of CJA_3703 (MLKTTLLFVVTALAEIIGCFLPYLWLRKGGSIWLLLPAALSLALFAWLLTLHPTASGRVYAAYGGVYVAVALLWLYWVDGVKLSAYDWAGAAVALLGMAIIAMGWQRS) , generate 3D homology models based on known structures of related proteins.

  • Compare predicted structural features, particularly the arrangement of transmembrane domains and potential functional sites.

  • Use molecular dynamics simulations to assess structural stability and potential conformational changes in different membrane environments.

• Genomic Context Analysis:

  • Examine the genomic neighborhood of CJA_3703 and its homologs in other bacteria.

  • Identify conserved gene clusters that might indicate functional associations or pathways involving these membrane proteins.

  • Apply comparative genomics techniques to correlate gene presence/absence patterns with specific metabolic capabilities across diverse bacterial species.

• Functional Complementation Studies:

  • Design experiments where homologs from different bacteria are expressed in a CJA_3703 knockout strain of C. japonicus.

  • Assess the ability of these homologs to restore wild-type phenotypes under various growth conditions.

  • Create chimeric proteins by swapping domains between CJA_3703 and its homologs to identify functionally important regions.

This comprehensive approach will provide insights into both conserved and species-specific aspects of UPF0060 family membrane proteins in the context of plant biomass degradation.

What methodologies can be employed to investigate potential protein-protein interactions involving CJA_3703 in the context of plant cell wall degradation pathways?

Investigating protein-protein interactions (PPIs) involving membrane proteins like CJA_3703 presents unique challenges due to their hydrophobic nature and membrane localization. Here are methodological approaches specifically tailored for this research question:

• Bacterial Two-Hybrid Systems Modified for Membrane Proteins:

  • Use BACTH (Bacterial Adenylate Cyclase Two-Hybrid) system, which is suitable for membrane protein interactions.

  • Design constructs that fuse different domains of the T18 and T25 fragments of adenylate cyclase to CJA_3703 and potential interaction partners from the C. japonicus proteome.

  • Screen for positive interactions by measuring cAMP production via β-galactosidase activity.

• In vivo Crosslinking Coupled with Mass Spectrometry:

  • Treat C. japonicus cells with membrane-permeable crosslinkers during growth on plant polysaccharides.

  • Purify CJA_3703 using the His-tag under denaturing conditions to maintain crosslinked complexes.

  • Identify crosslinked proteins using LC-MS/MS analysis and specialized software for crosslink identification.

  • Validate interactions using reciprocal pulldowns and co-immunoprecipitation.

• Proximity Labeling Techniques:

  • Generate fusion proteins of CJA_3703 with enzymes like BioID or APEX2.

  • Express these constructs in C. japonicus during growth on different plant polysaccharides.

  • Identify proteins in proximity to CJA_3703 through biotinylation followed by streptavidin pulldown and mass spectrometry.

• Co-evolution Analysis and Interactome Prediction:

  • Apply computational methods such as Direct Coupling Analysis (DCA) or GREMLIN to identify potential interaction partners based on co-evolutionary patterns.

  • Validate top predictions experimentally using techniques described above.

• Fluorescence-Based Interaction Assays:

  • Use Förster Resonance Energy Transfer (FRET) or Bimolecular Fluorescence Complementation (BiFC) with fluorescently tagged versions of CJA_3703 and candidate interaction partners.

  • Visualize interactions in vivo during bacterial growth on plant cell wall substrates.

These methodologies should be applied in the context of C. japonicus growth on plant polysaccharides to identify interactions relevant to the bacterium's plant cell wall degradation capabilities, which involve approximately 130 predicted glycoside hydrolases targeting major structural and storage plant polysaccharides .

How can recombinant CJA_3703 be integrated into synthetic biology applications for enhanced biomass degradation?

Leveraging recombinant CJA_3703 in synthetic biology applications for improved biomass degradation requires sophisticated design principles and methodological approaches:

These applications build upon the understanding that C. japonicus possesses an extensive range of glycoside hydrolases, lyases, and esterases for plant cell wall degradation , and that these capabilities might be enhanced through synthetic biology approaches incorporating membrane proteins like CJA_3703.