title: Expression of recombinant staphylokinase in the methylotrophic yeast Hansenula polymorpha.
abstract: BACKGROUND
  Currently, 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  The optimized production process described for rSAK-2 in Hansenula polymorpha provides an excellent, economically superior, manufacturing platform for a promising therapeutic fibrinolytic agent.
authors.affiliation: Minapharm Pharmaceuticals, Cairo, Egypt. mueller@minapharm.com
authors.forename: Manal
authors.initials: M
authors.lastname: Moussa
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Ibrahim
authors.forename: Maria
authors.initials: M
authors.lastname: El Ghazaly
authors.forename: Jan
authors.initials: J
authors.lastname: Rohde
authors.forename: Stefan
authors.initials: S
authors.lastname: Gnoth
authors.forename: Andreas
authors.initials: A
authors.lastname: Anton
authors.forename: Frank
authors.initials: F
authors.lastname: Kensy
authors.forename: Frank
authors.initials: F
authors.lastname: Mueller
mesh_terms: D000595:Amino Acid Sequence; D061249:Batch Cell Culture Techniques; D018533:Biomass; D019149:Bioreactors; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010843:Pichia / Q000254:growth & development / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013300:Streptokinase / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D013300:Streptokinase