Transthyretin (TTR) Recombinant Proteins are proteins produced using recombinant DNA technology that mimic or represent the naturally occurring transthyretin protein. Transthyretin is a plasma protein primarily responsible for transporting thyroid hormones and retinol-binding protein in the bloodstream.
- Gene Cloning and Expression:
- Gene Selection: The gene encoding human transthyretin (TTR) is identified and isolated from a cDNA library or genomic DNA.
- Vector Construction: The TTR gene is inserted into an expression vector that includes necessary regulatory elements (e.g., promoter, ribosome-binding site) to drive protein expression in a host cell.
- Host Cells: The recombinant vector is introduced into host cells (such as E. coli, yeast, insect cells, or mammalian cells) through transformation or transfection.
- Protein Expression:
- Induction Conditions: The host cells are cultured under conditions that promote the expression of the recombinant TTR protein. This often involves optimizing temperature, nutrient composition, and inducers.
- Protein Production: The TTR protein is synthesized and accumulates within the host cells or is secreted into the culture medium, depending on the expression system used.
- Protein Purification:
- Cell Harvesting: Cells or culture supernatants are collected, and the recombinant protein is extracted.
- Purification Techniques: The TTR protein is purified using methods such as affinity chromatography, ion-exchange chromatography, and size-exclusion chromatography. Tags included in the vector (e.g., His-tag) can facilitate purification.
- Refolding (if necessary): If the protein is expressed in an insoluble form (inclusion bodies), it may need to be refolded to achieve its functional conformation.
- Characterization and Validation:
- SDS-PAGE and Western Blotting: Used to confirm the size and identity of the recombinant TTR protein.
- Mass Spectrometry: Provides detailed information on the protein's structure and any post-translational modifications.
- Functional Assays: To evaluate the biological activity of the recombinant TTR, such as its ability to bind thyroid hormones or retinol-binding protein.
- Applications:
- Research: Recombinant TTR proteins are used to study TTR's role in physiological processes, such as hormone transport, and its involvement in diseases like amyloidosis.
- Diagnostics: These proteins can be used in assays to detect TTR levels or mutations associated with genetic disorders.
- Therapeutics: Recombinant TTR may be used as a therapeutic agent or as a basis for developing therapies for TTR-related diseases.
Transthyretin Recombinant Proteins play a critical role in understanding TTR-related functions and disorders, and they are valuable tools in both research and clinical applications.
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