Abstract
Human tissue-type plasminogen activator is one of the most important therapeutic proteins involved in the breakdown of blood clots following the stroke. A mutation was found at position 1541 bp (G514E) and the mutated form was cloned into the binary vector pTRAc-ERH. In silico analysis showed that this mutation might have no significant effect on the active site of the tissue plasminogen activator enzyme. Accordingly, zymography assay confirmed the serine protease activity of the mutated form and its derivatives. The expression of the mutated form was verified with/without co-agroinjection of the P19 gene silencing suppressor in both Nicotiana tabacum and N. benthamiana. The ELISA results showed that the concentration of the mutated form in the absence of P19 was 0.65% and 0.74% of total soluble protein versus 0.141% and 1.36% in the presence of P19 in N. benthamiana and N. tabacum, respectively. In N. tabacum, co-agroinjection of P19 had the synergistic effect and increased the mutated tissue plasminogen activator production two-fold higher. However, in N. benthamiana, the presence of P19 had the adverse effect of five-fold reduction in the concentration. Moreover, results showed that the activity of the mutated form and its derivatives was more than that of the purified commercial tissue plasminogen activator.
Highlights
Tissue plasminogen activator is a serine protease with a molecular mass of ~64 kDa1
Considering the high importance and expensiveness of Tissue plasminogen activator (tPA), the recombinant tPA has been expressed in a wide range of the different expression systems like bacteria, fungi, insect cells, transgenic animals, and plants[5]
A major drawback of this technique is the post-transcriptional gene silencing (PTGS) as a limiting factor for the production which could be overcame by the P19 protein, a plant virus encoded silencing suppressor protein[26,27]
Summary
The molecular docking of tPA and mtPA and plasminogen (PDB 4DCB) has been conducted using the HADDOCK web server) http://www.nmr.chem.uu.nl/haddock) to carry out comparative analysis between native and mutant forms. The pellet was resuspended in 25 ml of the cold sterilized solution (272 mM sucrose and 15% glycerol) and left for 5 minutes and centrifuged. The overnight culture was deposited by the centrifugation at 5,000 g for 15 min, suspended in an agroinjection buffer[41], and diluted to OD600 = 0.3. Supernatant was transferred to a fresh 1.5 ml microfuge tube after the centrifugation at 24,000 × g, 4 °C for 20 min and its total soluble protein concentration was measured by the Bradford technique[42]. The goat anti-rabbit IgG-HRP antibody (Sc-2030, USA) was added to blot as 1:2000 dilution for 1 h and washed as above with PBST for 10 min. Models Template Model 1 Model 2 Model 3 Model 4 Model 5 Model 6 Model 7 Model 8 Model 9 Model 10
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