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BMC Biotechnology

, 12:96

Microbial biotechnology


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 studys 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.

KeywordsStaphylokinaseHansenula polymorphaRecombinant proteinFermentationScale-upHTPElectronic supplementary materialThe online version of this article doi:10.1186-1472-6750-12-96 contains supplementary material, which is available to authorized users.

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Autor: ManalMoussa - MahmoudIbrahim - MariaEl Ghazaly - JanRohde - StefanGnoth - AndreasAnton - FrankKensy - FrankMueller


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