tPA Human
(a) Analysis by RP-HPLC.
(b) Analysis by SDS-PAGE.
Description
The tPA is purified by proprietary chromatographic techniques.
Product Specs
Product Science Overview
Tissue Plasminogen Activator (tPA) is a serine protease enzyme that plays a crucial role in the breakdown of blood clots. It is primarily found on the endothelial cells lining the blood vessels and is involved in the conversion of plasminogen to plasmin, the major enzyme responsible for clot breakdown. The recombinant form of this enzyme, known as Human Recombinant Tissue Plasminogen Activator (rtPA), has been developed for therapeutic use, particularly in the treatment of thrombotic diseases such as myocardial infarction, pulmonary embolism, and ischemic stroke .
The history of tPA dates back to the mid-20th century when researchers first identified and extracted a plasminogen activator from animal tissues. This enzyme was initially named fibrinokinase. The significant breakthrough came in 1982 when Genentech successfully produced tPA using recombinant DNA techniques . This advancement allowed for the large-scale production of tPA, making it widely available for clinical use.
Human tPA is composed of 527 amino acid residues and contains 17 disulfide bonds . It has a molecular weight of approximately 70 kDa in its single-chain form. The enzyme consists of five distinct structural domains:
- Finger Domain (F domain): Residues 4–50
- Epidermal Growth Factor-like Domain (E domain): Residues 50–87
- Two Kringle Domains (K1 and K2 domains): K1 (residues 87–176) and K2 (residues 176–256)
- Serine Protease Catalytic Domain (P domain): Residues 276–527
The binding of tPA to fibrin and the subsequent modulation of its protease activity are primarily regulated by the F and K2 domains. This binding is essential for the conversion of plasminogen to plasmin, which then degrades fibrin clots.
Recombinant tPA (rtPA) is used in the medical treatment of various thrombotic conditions. It is particularly effective in the management of acute ischemic stroke, where timely administration can significantly improve patient outcomes by dissolving the clot and restoring blood flow to the affected area of the brain . Other clinical applications include the treatment of myocardial infarction and pulmonary embolism.
The production of recombinant tPA involves the use of recombinant DNA technology. Initially, the cDNA encoding tPA is inserted into an expression vector, which is then introduced into a host cell line, such as Chinese Hamster Ovary (CHO) cells. These cells are cultured under conditions that promote the expression and secretion of tPA into the culture medium. The recombinant protein is then purified using various chromatographic techniques to obtain a highly pure and active form of tPA .
One of the main challenges in the production of recombinant tPA is achieving high yields of correctly folded and active protein. The presence of multiple disulfide bonds in tPA makes its expression and folding in prokaryotic systems like Escherichia coli particularly challenging. However, advancements in expression systems and purification techniques have improved the efficiency of tPA production .
Future research is focused on developing more efficient and cost-effective methods for producing recombinant tPA, as well as exploring its potential applications in other medical conditions. Additionally, efforts are being made to engineer tPA variants with improved stability and activity.
