Active Expression of Human Tissue Plasminogen Activator (t-PA) c-DNA from Pulmonary Metastases in the Methylotrophic Yeast Pichia Pastoris KM71H Strain

Abstract

Background: Human tissue-type plasminogen activator (t-PA) is a key protease of the trypsin family. It catalyzes the activation of zymogen plasminogen to the fibrin-degrading proteinase, plasmin, leading to digestion of fibrin clots. The recombinant enzyme produced by recombinant technology issued to dissolve blood clots in treatment of various human diseases such as coronary artery thrombosis, pulmonary embolism, acute ischemic stroke (AIS). Pichia pastoris expression system is a unique system for the production of high level of recombinant proteins. GS115 and KM71H are two kinds of Pichia pastoris strains whilst production of recombinant proteins in these strains is not predictable. The aim of the study was evaluation of t-PA expression in KM71H strains. Methods: In this study, the cDNA of the t-PA gene was amplified by PCR, sequenced and cloned into Pichia pastoris KM71H host strain using pPICZalphaA expression vector that allows methanol-induced expression and secretion of the protein. Results: Dot blotting results confirmed the presence oft-PA in the cell supernatant. Western blotting test revealed the approximate size of 70 KDa for recombinant t-PA. Quantitative ELISA experiment showed 810 μg/L of t-PA in the supernatant samples. Zymography analysis confirmed the proteolytic activity and biological function of the expressed recombinant t-PA. Conclusions: Correspondingly, Pichia pastoris KM71H is an appropriate strain for production of active recombinant protein.

Keywords: Tissue plasminogen activator; Pichia pastoris; Fibrin; recombinant proteins.

Figure 1

The Parts of the pPICZalphaA Shuttle Vector Multiple Cloning Sites. As seen, the Kex2 signal cleavage site presented in the middle of XhoI restriction sites.

Figure 2

The Results Related to Restriction Map Preparation from Plasmid Constructs via SacI Enzyme. Line 1: 1 kb DNA marker; lines 2: corresponding to undigested plasmid; lines 3: corresponding to digested plasmid. The presence of 2992 bp and 2208 bp bands after digestion of recombinant construct proved the correct orientation of t-PA in pPICZalphaA.

Figure 3

Dot Blotting (A) and western blotting (B) analysis results. A: The lane 1: Positive control (t-PA standard, Actylase); lane 2-4 related to supernatants of time points 96 (4 days), 120 (5 days), and 144 (6 days) respectively; lane 5: Negative control (The supernatant of Pichia pastoris KM71H without t-PA gene). B: M: Protein marker; lane 1: Positive control (t-PA standard, Actylase); lane 2: the supernatant of recombinant Pichia pastoris KM71H; lane 3: Negative control (The supernatant of Pichia pastoris KM71H without t-PA gene).

Figure 4

The Standard Curve Related to Concentrations of Standard Human t-PA against of A450. According to This Curve, the Quantity of Extracellular t-PA Recombinant Protein (1/1,000) Calculated 810.79 µg/L.

Figure 5

The Result of Zymography Test. The lane 1: Positive control (t-PA standard, Actylase); lane 2 and 3 related to unconcentrated and concentrated supernatants of time point 120 (5 days) respectively; lane 4 and 5 related to unconcentrated and concentrated supernatants of time point 144 (6 days) respectively; lane 6: related to boiled supernatant of time point 144 as negative control.