Recombinant Human Cortexin-1 (CTXN1)

What is Cortexin-1 (CTXN1) and what is its basic structure?

Cortexin-1 is a small protein consisting of 82 amino acids with a molecular weight of approximately 9.0 kDa . The full amino acid sequence is: "MSATWTLSPE PLPPSTGPPV GAGLDAEQRT VFAFVLCLLV VLVLLMVRCV RILLDPYSRM PASSWTDHKE ALERGQFDYA LV" . Based on this sequence analysis, CTXN1 appears to contain a transmembrane domain, suggesting its involvement in membrane-associated functions in neuronal cells.

What is the putative function of CTXN1 in neural development?

According to current understanding, CTXN1 may mediate extracellular or intracellular signaling of cortical neurons during forebrain development . This function has been computationally predicted (as indicated by the ECO:0000250 tag in databases) rather than extensively experimentally verified. Given its expression in cortical neurons, it likely plays a role in neuronal signaling pathways that are essential for proper brain development.

What expression systems are recommended for producing recombinant CTXN1?

HEK-293 cells have been successfully used to express recombinant human CTXN1 . This mammalian expression system provides appropriate post-translational modifications for the human protein. For optimal results, the recombinant protein can be produced with an Fc tag to facilitate purification via affinity chromatography. The optimized expression system ensures reliability for producing intracellular, secreted, and transmembrane proteins .

How can researchers effectively design experiments to investigate CTXN1's role in neuronal signaling?

When investigating CTXN1's role in neuronal signaling, consider these approaches:

• Gene expression manipulation studies: Implement overexpression, knockdown, or knockout strategies in appropriate neuronal models.

• Protein interaction studies: Perform co-immunoprecipitation followed by mass spectrometry to identify binding partners.

• Functional assays: Use calcium imaging or electrophysiology to assess effects on neuronal activity.

• In vivo models: Develop and characterize transgenic animals with altered CTXN1 expression.

• Colocalization studies: Examine CTXN1 expression alongside other markers such as Cart (Cocaine and Amphetamine Regulated Transcript) which has been found in Calbindin-immunoreactive neurons .

What approaches are recommended for studying CTXN1's potential involvement in neurodevelopmental processes?

To investigate CTXN1's role in neurodevelopment:

• Temporal expression analysis: Track CTXN1 expression during different developmental stages.

• Conditional knockout models: Use inducible systems to manipulate CTXN1 expression at specific developmental timepoints.

• Cell-type specific analysis: Employ techniques like those used in the analysis of Cart-expressing neurons (stereotactic injections with viral constructs in Slc17a6::Cre or Slc32a1::Cre mice) .

• Neuronal differentiation assays: Assess the impact of CTXN1 manipulation on processes such as neuronal migration, axon guidance, and synaptogenesis.

• Transcriptomic analysis: Use approaches similar to those described for analyzing gene expression in the nervous system to identify CTXN1 co-expression networks .

How might CTXN1 function relate to neuronal health and disease?

Based on its putative role in neuronal signaling:

• Co-expression analysis: Determine if CTXN1 is co-expressed with genes critical for neuronal health and viability, as identified in gene expression network analyses .

• Disease models: Investigate CTXN1 expression in models of neurodevelopmental or neurodegenerative disorders.

• Genetic association studies: Examine potential links between CTXN1 variants and neurological conditions.

• Cell stress responses: Assess how CTXN1 expression responds to neuronal stressors such as those that activate c-fos expression .

What are the recommended protocols for purifying recombinant CTXN1?

For optimal purification of recombinant CTXN1:

• Expression system: Use HEK-293 cells to express the protein with an Fc tag .

• Purification method: Employ one-step affinity chromatography utilizing the Fc tag.

• Quality control: Verify purity (>90%) using Bis-Tris PAGE, anti-tag ELISA, Western Blot, and analytical SEC (HPLC) .

• Storage: Store at -80°C and avoid repeated freeze-thaw cycles .

What considerations are important when designing functional studies with recombinant CTXN1?

When planning functional studies:

• Protein conformation: Ensure the recombinant protein maintains its native structure.

• Tag interference: Consider whether the Fc tag might interfere with protein function; if necessary, explore tag removal options.

• Positive controls: Include appropriate controls to validate experimental systems.

• Concentration range: Test a range of concentrations to identify dose-dependent effects.

• Experimental validation: As noted in product information, while the recombinant protein is expected to work for functional studies, validation is necessary as this has not been extensively tested .

How can researchers effectively characterize CTXN1 expression in neural tissues?

For thorough characterization of CTXN1 expression:

• Immunohistochemistry: Use validated antibodies against CTXN1 in tissue sections.

• In situ hybridization: Apply techniques similar to those used for Calb1 expression analysis in mouse brain .

• Region-specific analysis: Define regions of interest (ROI) that encompass CTXN1-expressing cells, similar to approaches used for other neuronal markers .

• Co-expression studies: Identify co-expression with neurotransmitter markers using approaches like those employed for Cart expression (using Slc17a6::Cre, Slc32a1::Cre, and Slc6a5-GFP::Cre mice) .

• Single-cell analysis: Consider single-cell transcriptomics to identify cell-type specific expression patterns.

CTXN1 Protein Characteristics

CTXN1 Sequence Information

What are the current knowledge gaps regarding CTXN1?

Despite available recombinant protein resources, significant knowledge gaps exist:

• Precise signaling mechanisms: The specific molecular pathways through which CTXN1 influences neuronal development remain poorly characterized.

• Structural information: Detailed three-dimensional structure of CTXN1 requires further investigation.

• Expression patterns: Comprehensive mapping of CTXN1 expression across brain regions and developmental stages is needed.

• Functional validation: Experimental confirmation of computationally predicted functions is necessary.

• Disease relevance: Potential roles in neurological disorders need systematic exploration.

What emerging research approaches could advance our understanding of CTXN1?

To address current limitations in CTXN1 research:

• Network analysis approaches: Apply co-expression network analysis methods similar to those used for other neuronal genes .

• CRISPR-based gene editing: Generate precise modifications to study structure-function relationships.

• Organoid models: Utilize brain organoids to study CTXN1 in a developmentally relevant context.

• Multi-omics integration: Combine transcriptomic, proteomic, and functional data to build comprehensive models of CTXN1 function.

• Cross-species comparison: Compare CTXN1 function across species to identify evolutionarily conserved roles.