Development of a streptokinase expression platform using the native signal sequence of the protein with internal repeats 1 (PIR1) in P. pastoris: Gene dosage optimization and cell retention strategies

Abstract Streptokinase is a therapeutically important thrombolytic agent which is toxic to its heterologous expression hosts and also prone to proteolytic degradation. In the current study, its secretory expression was targeted in P. pastoris using the native protein with internal repeats 1 (PIR1) secretion signal where the optimization of streptokinase gene dosage resulted in an increase in the volumetric product yield from 339.69 to 1206.48 mg/L. The cell retention strategy was applied during the induction phase to maintain sustained productivity where up to a 2.4 fold enhancement in the overall product yield was achieved at shake flask to a level of 3049.53 mg/L. Moreover, the cell recycling approach prevented the streptokinase proteolytic degradation and also helped in achieving a high specific product yield (Y P/X ) in a range of 167.65–176.44 mg/g DCW even after five cycles. Similarly, the cell recycling strategy was also employed using S. cerevisiae α-mating factor (α-MF) signal sequence which also resulted in improved product stability and volumetric product concentration confirming its wide applicability in obtaining enhanced product quality and yield. The recombinant streptokinase produced using PIR1 signal sequence was biologically active possessing a specific activity of 62,782.40 IU/mg and was predicted to contain 10.45% α-helix and 29.09% β-sheet.