Recombinant Human Titin (TTN) is expressed in E. coli, covering the amino acid region 5398-5604 of Isoform 6. This protein carries an N-terminal 6xHis tag, which helps with purification and detection. SDS-PAGE analysis shows the protein appears to reach purity levels above 90%. The recombinant protein is produced under stringent conditions to keep endotoxin levels low, which should make it suitable for various research applications.
Titin plays a crucial role in muscle physiology - it's known for its involvement in muscle contraction and elasticity. As the largest protein in the human body, titin contributes to the structural integrity and function of the sarcomere, the basic unit of muscle contraction. Research on titin may be essential for understanding muscular disorders and the biomechanics of how muscles work.
Potential Applications
Note: The applications listed below are based on what we know about this protein's biological functions, published research, and experience from experts in the field. However, we haven't fully tested all of these applications ourselves yet. We'd recommend running some preliminary tests first to make sure they work for your specific research goals.
1. Protein-Protein Interaction Studies
This recombinant TTN fragment (5398-5604aa) could be used to investigate specific protein interactions within the sarcomere structure. The N-terminal 6xHis tag allows for purification and immobilization in pull-down assays to identify binding partners. Co-immunoprecipitation experiments and surface plasmon resonance studies might help characterize the binding kinetics and specificity of interactions involving this particular titin domain. Studies like these would likely contribute to understanding the molecular mechanisms of sarcomere assembly and maintenance.
2. Antibody Development and Validation
The purified recombinant protein fragment serves as what appears to be an ideal antigen for generating specific antibodies against this region of human titin. The high purity (>90%) should minimize cross-reactivity during immunization protocols. Generated antibodies can be validated through ELISA, Western blot, and immunofluorescence techniques with this recombinant protein as a positive control. This application seems particularly valuable given titin's large size - region-specific antibodies are essential for studying distinct functional domains.
3. Structural and Biophysical Characterization
The recombinant TTN fragment can be subjected to various biophysical analyses to understand the structural properties of this specific domain region. Techniques such as circular dichroism spectroscopy, dynamic light scattering, and analytical ultracentrifugation may provide insights into secondary structure, oligomerization state, and stability. The 6xHis tag makes protein purification to homogeneity easier for these sensitive analytical methods. Such characterization would likely contribute to the broader understanding of titin's modular architecture.
4. In Vitro Binding Assays
The His-tagged protein fragment can be used in enzyme-linked immunosorbent assays (ELISA) and other plate-based binding studies to screen for small molecule interactions or validate binding of other sarcomeric proteins. The tag allows for oriented immobilization on nickel-coated surfaces, which should ensure consistent protein presentation. This application could support drug discovery efforts targeting titin-related pathways. It might also help validate computational predictions of protein-ligand interactions within this specific domain region.
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