Recombinant Human Vezatin (VEZT)

What is Vezatin (VEZT)?

Vezatin (VEZT) is an integral membrane protein found in adherens junctions that facilitates cell-cell adhesion. According to structural analyses, Vezatin exists in two main isoforms in mice: long (88 kDa) and short (71 kDa), which differ in their C-terminal regions. These isoforms have been detected in mouse pre-implantation embryos, suggesting important developmental roles . The protein contains transmembrane domains that anchor it within the cell membrane, positioning it ideally for its role in cellular adhesion processes.

What is the molecular structure of Human Vezatin?

Human Vezatin is characterized by either one or two transmembrane domains, depending on the prediction method used. Multiple membrane-topology prediction programs reveal different possible configurations: MEMSAT and SOSUI predict two transmembrane domains (approximately at amino acids 134-158 and 166-188) with both N-terminal and C-terminal regions facing the cytoplasm, while TMHMM suggests a single transmembrane domain (amino acids 149-182) . This structural uncertainty highlights the need for experimental determination of Vezatin's precise topology, which could significantly impact understanding of its function.

What cellular functions has Vezatin been implicated in?

Vezatin functions primarily at adherens junctions, critical structures for tissue integrity and cellular communication. Studies in engineered mouse models lacking Vezatin in sensory cells of the inner ear have demonstrated its importance in hearing function . The protein's localization at cell-cell junctions suggests roles in maintaining epithelial barriers, tissue morphogenesis, and potentially in mechano-transduction pathways. These functions position Vezatin as an important molecule in development, tissue homeostasis, and sensory perception.

What expression systems are most effective for producing recombinant human Vezatin?

For recombinant human Vezatin production, researchers should consider multiple expression systems based on experimental needs. Eukaryotic expression systems, including mammalian cells, are often preferred for membrane proteins like Vezatin to ensure proper folding and post-translational modifications. Available options include "eukaryotic plasmid vectors, eukaryotic viral vectors, prokaryotic plasmids, bacteriophage vectors, shuttle vectors, mini-chromosomes, and various artificial chromosomes" . For stable integration into host genomes, plasmid vectors that integrate by non-homologous recombination offer particular advantages for long-term expression studies .

How can expression levels of recombinant human Vezatin be enhanced?

Expression levels of recombinant human Vezatin can be significantly enhanced by incorporating specific recombinant expression vector elements (rEVEs). Research has identified sequences termed "ARM1" (SEQ ID NO:1) and "ARM2" (SEQ ID NO:2) that, when incorporated into expression vectors, enhance protein production compared to vectors lacking these elements . Additionally, implementation of the DHFR-methotrexate amplification system can further increase protein yield by selecting for cells with multiple vector copies, achieving methotrexate resistance while maintaining elevated protein expression even after selective pressure is removed .

What host cell considerations are important when expressing recombinant Vezatin?

Host cell selection is critical for successful Vezatin expression. Both eukaryotic and prokaryotic host cells can be employed, though the membrane protein nature of Vezatin typically favors eukaryotic systems. Preferred eukaryotic hosts include "mammalian host cells, plant host cells, fungal host cells, eukaryotic algal host cells, protozoan host cells, insect host cells, and fish host cells" . When using the DHFR-methotrexate amplification system, researchers should monitor methotrexate adaptation, as vectors containing rEVEs demonstrate improved host cell survivability and growth rates in methotrexate-containing media compared to vectors lacking these elements .

What purification strategies work best for recombinant human Vezatin?

Purification of recombinant human Vezatin requires specialized approaches due to its membrane-embedded nature. An effective protocol begins with careful cell lysis in the presence of membrane-protecting detergents, followed by differential centrifugation to isolate membrane fractions. Subsequent solubilization with mild detergents like n-dodecyl-β-D-maltoside (DDM) or digitonin helps maintain protein structure. Affinity chromatography utilizing engineered tags (His, FLAG, etc.) allows for selective binding and elution of the target protein. Size exclusion chromatography provides final purification and confirms protein homogeneity. Throughout purification, protein stability should be monitored by activity assays and structural validation techniques.

How can researchers verify proper folding and functionality of purified Vezatin?

Verification of properly folded and functional Vezatin involves multiple complementary approaches. Circular dichroism spectroscopy can confirm secondary structure elements characteristic of properly folded membrane proteins. Thermal stability assays evaluate protein stability and can screen for optimal buffer conditions. For functional validation, researchers should develop binding assays with known interaction partners. Reconstitution into artificial membrane systems (liposomes, nanodiscs) followed by functional assays provides the gold standard for verification. Additionally, cell-based complementation assays where the recombinant protein rescues function in Vezatin-deficient cells offer powerful functional validation.

What techniques are recommended for studying Vezatin's protein-protein interactions?

For studying Vezatin's protein-protein interactions, researchers should employ techniques that preserve the native membrane environment. Proximity labeling methods like BioID or APEX2 can identify interaction partners in living cells. Crosslinking mass spectrometry captures transient interactions and provides structural information. Co-immunoprecipitation from membrane fractions with appropriate detergents can maintain relevant interactions. For quantitative interaction measurements, microscale thermophoresis or surface plasmon resonance with reconstituted protein in nanodiscs is recommended. Computational prediction tools can guide these experimental approaches by identifying potential interaction partners based on structural and evolutionary data.

How can researchers overcome low expression yields of recombinant human Vezatin?

Low expression yields of recombinant human Vezatin can be addressed through systematic optimization strategies. Incorporate rEVEs like ARM1 or ARM2 sequences into the expression vector, as these have been demonstrated to enhance recombinant protein expression levels . Implement the DHFR-methotrexate amplification system with methotrexate concentrations ranging from "20 nM to 500 nM" or even "5 nM to 10 μM" to select for cells with amplified gene expression . Consider codon optimization for the host system and carefully evaluate different promoter strengths. Testing multiple truncation constructs may identify better-expressing variants while still retaining functional domains.

What strategies help prevent Vezatin aggregation during expression and purification?

Preventing Vezatin aggregation requires careful management of the protein's hydrophobic transmembrane regions. During expression, lower induction temperatures (16-25°C) slow protein synthesis, allowing more time for proper membrane insertion. Adding chemical chaperones to culture media can stabilize folding intermediates. During purification, selection of appropriate detergents is critical—screening multiple detergent types and concentrations identifies optimal solubilization conditions. Addition of cholesterol or specific lipids can stabilize native conformation. Using fusion partners like GFP can improve solubility and provide a convenient method to monitor aggregation state. Finally, inclusion of glycerol (5-10%) in all buffers helps maintain protein stability throughout the purification process.

How can researchers troubleshoot issues with Vezatin's membrane topology during expression?

Troubleshooting membrane topology issues requires strategic approaches given Vezatin's complex structure with predicted transmembrane domains at approximately amino acids 134-158 and 166-188 . Implement topology mapping using accessibility labeling with membrane-impermeable reagents. Create fusion constructs with topology reporter proteins (GFP, alkaline phosphatase) at different positions. Compare results from multiple prediction algorithms (MEMSAT, SOSUI, TMHMM) to guide construct design . Consider expressing individual domains separately to validate their localization. Perform protease protection assays on intact membrane preparations to experimentally determine which regions are protected. Cross-validate findings using multiple approaches, as prediction programs have shown inconsistent results for Vezatin's topology .

How is Vezatin being used in disease model research?

Vezatin's role in disease models centers on its critical function in cell adhesion and tissue integrity. Mouse models with conditional Vezatin knockout in sensory cells of the inner ear have been engineered to study its role in hearing disorders . This approach can be extended to other tissues where adherens junctions play important roles. Recombinant DNA technology enables the creation of these disease models by precisely controlling gene expression in target tissues . The technology allows researchers to study how Vezatin dysfunction contributes to pathological processes and potentially identify therapeutic targets for conditions involving cell adhesion defects.

What gene editing approaches are most effective for studying Vezatin function?

Gene editing to study Vezatin function benefits from recombinant DNA technology's ability to create precise genetic modifications . CRISPR-Cas9 systems offer the most versatile approach, enabling knockout, knockin, or domain-specific mutations. For conditional control, systems combining Cre-LoxP with CRISPR allow tissue-specific deletion. Homology-directed repair using recombinant templates permits introduction of specific mutations or reporter tags. Base editing techniques can introduce point mutations without double-strand breaks. When designing editing strategies, researchers should consider potential compensatory mechanisms from related proteins and plan appropriate controls. Phenotypic characterization should include both cellular and tissue-level analyses to fully understand Vezatin's function.

How can computational approaches enhance experimental research on Vezatin?

Computational approaches significantly enhance Vezatin research through multiple avenues. Structure prediction algorithms help model Vezatin's conformation, particularly important given the conflicting predictions from different topology programs (MEMSAT, SOSUI, TMHMM) . Molecular dynamics simulations can model behavior within membrane environments. Sequence analysis across species identifies conserved domains likely critical for function. Protein-protein interaction networks predict functional relationships and potential binding partners. Systems biology approaches integrate Vezatin into larger cellular pathways. These computational tools guide experimental design by generating testable hypotheses and helping interpret experimental results within a broader biological context, ultimately accelerating discovery while reducing resource expenditure.