In silico and in vivo analyses of the mutated human tissue plasminogen activator (mtPA) and the antithetical effects of P19 silencing suppressor on its expression in two Nicotiana species

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.

Figure 1

Schematic representation of the various molecular forms of t-PA.

Figure 2

Schematic overview of the pMA2 construct (pTRAc-tPA-ERH).

Figure 3

Surface representation of the interaction between the native form of tPA and plasminogen.

Figure 4

Surface representation of the interaction between the mutated form of tPA and plasminogen.

Figure 5

Ligplot diagram of the interactions at the protein–protein interface in (a) native form, (b) mutant form of tPA and plasminogen.

Figure 6

RT-PCR amplification of tissue plasminogen activator on RNA extracted from agroinjected leaves.

Figure 7

Dot blot analysis using tPA-specific antibody in two replicates.

Figure 8

Western blot analysis with tPA-specific antibody.

Figure 9

Transient expression of recombinant protein mtPA in agroinjected leaves of Nicotiana species.

Figure 10

Zymography assay on extracted protein of injectd leaves.

Figure 11

Quantitive data from zymogram and western blot comparing the commercial tPA vs. the mutated tPA.