Tissue-type plasminogen activator (t-PA) is an enzyme that is involved in breaking down blood clots. In humans, t-PA is found in the blood where it serves as the activator of the fibrinolytic system. It converts plasminogen, a zymogen, to plasmin, an enzyme that functions to break down fibrin (a protein involved in the formation of blood clots) .
t-PA is a serine protease comprised of a single polypeptide chain containing 527 amino acids. It has a molecular weight of 72 kDa . This protein contains five functional domains: two kringle domains (K1 and K2), a C-terminal serine protease domain (S), an epidermal growth factor-like domain (EGF) domain, and a finger domain (F) . In blood, t-PA is the physiological activator of the fibrinolytic system. As stated previously, it converts plasminogen to plasmin. Plasmin then hydrolyzes t-PA into a two-chain form by cleaving the Arg 275-Ile 276 peptide bond . t-PA is a thrombolytic agent, meaning it dissolves blood clots, and it's recombinant forms are used pharmacologically for treating acute ischemic stroke, acute myocardial infarction, and pulmonary embolism .
Recombinant tissue-type plasminogen activator (rt-PA)Edit
The recombinant forms of tissue-type plasminogen activator (rt-PA) are manufactured by the pharmaceutical industry for the treatment of acute ischemic stroke (stroke) myocardial infarction (heart attack) and pulmonary embolism. Drugs such as Activase(alteplase), Retavase (reteplase), and Tenecteplase are all forms of recombinantly-derived t-PA.
Recombinant forms of t-PA (rt-PAs) are engineered to have slightly different structures than naturally occuring t-PAs to both prevent their blood clot-dissolving action from being inhibited, and to increase their efficacy and plasma half-lives as pharmacuetical agents. In blood plasma, t-PAs are inhibited by specific plasminogen activator inhibitors (PAIs), mainly PAI-1, that rapidly inhibit both the single chain and two-chain forms of t-PA. Recombinant forms are engineered to prevent this inhibition from occuring. Back in the late 1980's researchers identified specific amino acid residues in t-PA, the region of the protein consisting of residues 269 to 304, that are involved in its interaction with the inhibitor PAI-1 but not in its interaction with its substrate plasminogen. Thus, these researchers were able to induce mutations in this region of the t-PA protein by site-directed mutagenesis that successfully rendered rt-PA resistant to purified PAI-1 . From 1989 to the present, many research groups and private biotech and pharmaceutical companies have enginerred their own forms of rt-PA and rt-PA derivatives that vary in both the amino acid sequence of the region comprising residues 296 to 304, with respect to mutations and deletions, and in the functional domains they contain (Figure 1) . One example of an rt-PA derivative is K2S, a 39 kD derivative of t-PA containing only the kringle 2 (K2) and serise protease (S) domains. K2S remains active in the plasma for a longer amount of time (has an increased plasma half-life) and exhibits reduced hepatic clearance (the loss of a drug during its passage through the liver) compared to other forms of rt-PA .
Recombinant DNA ConstructEdit
"Fig. 1 (diagram pictured above)| Map of the expression construct used for transfection of L. tarentolae. Abbreviations: 50 ssu and 30 ssu are regions of the small subunit of L. tarentolae rRNA gene for homologous recombination into the host chromosome following linearization of the expression plasmid with SwaI. 0.4k-IR camBA, 1.4k-IR camCB and 1.7k-IR are optimized gene-flanking non-translated regions providing the splicing signals for posttranscriptional mRNA processing for expression of target and marker genes in the L. tarentolae. SP designates the signal peptide of L. mexicana secreted acid phosphatase. Hyg marker gene for selection with hygromycin." 
Target Cells used for ExpressionEdit
Several different types of cells lines are used for expression of rt-PA including various E.coli cells lines (XL-1 Blue, K-12, etc.), Chinese hampster ovary (CHO) cells, and Leishmania tarentolae (L. tarentolae), a non-pathogenic lizard-infecting species of the Leishmania trypanosomatid protozoa. L. tarentolae is used as a system for producing K2S, because when the K2S sequence is inserted into E. coli it leads to the formation of inclusion bodies that are inactive and must be subsequently purified and refolded in order to become active. The formation of such inclusion bodies does not occur when the K2S sequence in inserted into and expressed by L. tarentolae cells .
There are many ways in which recombinant proteins, or proteins in general, can be purified. The way in which the truncated rt-PA protein (rK2S), discussed here, which was expressed in L. tarentolae was purified was by affinity chromatography. The fractions were analyzed by SDS-PAGE, and the investigators found a desired band at 40 kDa, which was slightly higher than the native mature protein due to the presence of a hexa-his-tag sequence (Figure 2). They used western blotting to detect the presence of rK2S in the culture supernatants and in the fractions of purification (Figure 2). The researchers noted that the sharp clear zones on the blue background of the zymography gel indicated the activity of rK2S serene protease, and this was observed only in the culture supernatant of transformed L. tarentolae (Figure 3). Furthermore, the researchers showed that there was enzymatic activity of 931 i.u./ml of the culture supernatant of transformed L. tarentolae with secretory expression vector pF4splmsapx1.4hyg containing the K2S gene. They then calculated the specific activity of purified rK2S to be 7.43 U/mg of protein .
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