Expression of recombinant staphylokinase in the methylotrophic yeast Hansenula polymorpha

Abstract

BackgroundCurrently, the two most commonly used fibrinolytic agents in thrombolytic therapy are recombinant tissue plasminogen activator (rt-PA) and streptokinase (SK). Whereas SK has the advantage of substantially lower costs when compared to other agents, it is less effective than either rt-PA or related variants, has significant allergenic potential, lacks fibrin selectivity and causes transient hypotensive effects in high dosing schedules. Therefore, development of an alternative fibrinolytic agent having superior efficacy to SK, approaching that of rt-PA, together with a similar or enhanced safety profile and advantageous cost-benefit ratio, would be of substantial importance. Pre-clinical data suggest that the novel fibrinolytic recombinant staphylokinase (rSAK), or related rSAK variants, could be candidates for such development. However, since an efficient expression system for rSAK is still lacking, it has not yet been fully developed or evaluated for clinical purposes. This study’s goal was development of an efficient fermentation process for the production of a modified, non-glycosylated, biologically active rSAK, namely rSAK-2, using the well-established single cell yeast Hansenula polymorpha expression system.ResultsThe development of an efficient large scale (80 L) Hansenula polymorpha fermentation process of short duration for rSAK-2 production is described. It evolved from an initial 1mL HTP methodology by successive scale-up over almost 5 orders of magnitude and improvement steps, including the optimization of critical process parameters (e.g. temperature, pH, feeding strategy, medium composition, etc.). Potential glycosylation of rSAK-2 was successfully suppressed through amino acid substitution within its only N-acetyl glycosylation motif. Expression at high yields (≥ 1g rSAK-2/L cell culture broth) of biologically active rSAK-2 of expected molecular weight was achieved.ConclusionThe optimized production process described for rSAK-2 in Hansenula polymorpha provides an excellent, economically superior, manufacturing platform for a promising therapeutic fibrinolytic agent.

Comparison of expression optimization of new derivative of staphylokinase (SAK‐2RGD‐TTI) with the rSAK

“…The quantity of the purified protein was 420 μg/mL for SAK‐2RGD‐TTI‐2, with a purification factor of 2.37 and a relatively low yield of 51.24% due to exceeding the capacity of the Ni‐IDA resin and losing significant amount of loaded protein namely as flow‐through (Table ). That was consistent with previous studies cited that staphylokinase or its multifunctional derivatives were the main protein in the supernatant . The SAK‐2RGD‐TTI increased lysis zone on agar‐plasma plate up to average 109 mm 2 (420 μg/mL) in a dose‐dependent manner (Figure c).…”

“…In that study, an additional “smear‐like” band between 20 and 25 kDa, indicating glycosylated rSAK‐1 in addition to a nonglycosylated band of 16 kDa was visualized. Further, substitution of amino acid of threonine with alanine in N‐glycosylation recognition site resulted in the expression of only nonglycosylated rSAK‐2 variant …”

“…That was consistent with previous studies cited that staphylokinase or its multifunctional derivatives were the main protein in the supernatant. 9,18,27,42,53 The SAK-2RGD-TTI increased lysis zone on agar-plasma plate up to average 109 mm 2 (420 μg/mL) in a dose-dependent manner ( Figure 11c). According to the well diffusion method using streptokinase T A B L E 2 Purification procedure of recombinant SAK-2RGD-TTI protein from the supernatant of P. pastoris strain GS115.…”

Efficient CRISPR–Cas9 mediated multiplex genome editing in yeasts

“…It has been an important organism for studies on methanol utilization, autophagy, peroxisome biogenesis and nitrate assimilation [ 6 ]. One attractive property of O. polymorpha is able to integrate up to 100 copies of target gene into the genome mediated by non-homologous end joining (NHEJ), which can be used to highly express heterologous genes and synthesize various biotechnology products [ 1 , 4 , 7 , 8 ]. Furthermore, it can synthesize glycoproteins with human compatible oligosaccharides [ 9 , 10 ].…”

The pharmaceutics from the foreign empire: the molecular pharming of the prokaryotic staphylokinase in Arabidopsis thaliana plants

“…However, the protein purification process may appear to be more difficult, and thus economically less feasible than expected (Prasad et al 2010 ). During the SAK recovery its activity may drastically decrease, for example due to the solubilisation process, thus both the bacterial and yeast systems for the staphylokinase production is still under investigation (Kotra et al 2013 ; Moussa et al 2012 ). Plants seemed to be an alternative and far more attractive system of staphylokinase production.…”