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Expression of recombinant staphylokinase in the methylotrophic yeast Hansenula polymorpha.

Moussa M, Ibrahim M, El Ghazaly M, Rohde J, Gnoth S, Anton A, Kensy F, Mueller F - BMC Biotechnol. (2012)

Bottom Line: 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.Potential glycosylation of rSAK-2 was successfully suppressed through amino acid substitution within its only N-acetyl glycosylation motif.The optimized production process described for rSAK-2 in Hansenula polymorpha provides an excellent, economically superior, manufacturing platform for a promising therapeutic fibrinolytic agent.

View Article: PubMed Central - HTML - PubMed

Affiliation: Minapharm Pharmaceuticals, Cairo, Egypt. mueller@minapharm.com

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.

Conclusion: The optimized production process described for rSAK-2 in Hansenula polymorpha provides an excellent, economically superior, manufacturing platform for a promising therapeutic fibrinolytic agent.

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Related in: MedlinePlus

Amino acid sequences of wt-SAK and SAK-variants [rSAK-1 (= THR 174) and rSAK-2]. The amino acid sequence of each of the different SAK molecules, highlighting the differences in their primary structures (blue), is shown. Red: Potential N-linked glycosylation-site motif. Red underlined: Amino acid exchange Thr-30 → Ala-30.
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Figure 1: Amino acid sequences of wt-SAK and SAK-variants [rSAK-1 (= THR 174) and rSAK-2]. The amino acid sequence of each of the different SAK molecules, highlighting the differences in their primary structures (blue), is shown. Red: Potential N-linked glycosylation-site motif. Red underlined: Amino acid exchange Thr-30 → Ala-30.

Mentions: Initial trials for the correct protein expression productivity of rSAK-1 and rSAK-2 (see Figure 1) transformants (strains) were performed in shake flasks using YPG medium. As shown in Western blots of culture supernatants from the rSAK-2 strain (Figure 2a), a single well-defined band at around 16 kDa that migrated parallel to that of E. coli-produced rSAK-1 (ThromboGenics NV) is present. The rSAK-1 strain, however, produced an additional ‘smear-like’ band between 20 kDa and 25 kDa, indicating glycosylated rSAK-1.


Expression of recombinant staphylokinase in the methylotrophic yeast Hansenula polymorpha.

Moussa M, Ibrahim M, El Ghazaly M, Rohde J, Gnoth S, Anton A, Kensy F, Mueller F - BMC Biotechnol. (2012)

Amino acid sequences of wt-SAK and SAK-variants [rSAK-1 (= THR 174) and rSAK-2]. The amino acid sequence of each of the different SAK molecules, highlighting the differences in their primary structures (blue), is shown. Red: Potential N-linked glycosylation-site motif. Red underlined: Amino acid exchange Thr-30 → Ala-30.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539880&req=5

Figure 1: Amino acid sequences of wt-SAK and SAK-variants [rSAK-1 (= THR 174) and rSAK-2]. The amino acid sequence of each of the different SAK molecules, highlighting the differences in their primary structures (blue), is shown. Red: Potential N-linked glycosylation-site motif. Red underlined: Amino acid exchange Thr-30 → Ala-30.
Mentions: Initial trials for the correct protein expression productivity of rSAK-1 and rSAK-2 (see Figure 1) transformants (strains) were performed in shake flasks using YPG medium. As shown in Western blots of culture supernatants from the rSAK-2 strain (Figure 2a), a single well-defined band at around 16 kDa that migrated parallel to that of E. coli-produced rSAK-1 (ThromboGenics NV) is present. The rSAK-1 strain, however, produced an additional ‘smear-like’ band between 20 kDa and 25 kDa, indicating glycosylated rSAK-1.

Bottom Line: 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.Potential glycosylation of rSAK-2 was successfully suppressed through amino acid substitution within its only N-acetyl glycosylation motif.The optimized production process described for rSAK-2 in Hansenula polymorpha provides an excellent, economically superior, manufacturing platform for a promising therapeutic fibrinolytic agent.

View Article: PubMed Central - HTML - PubMed

Affiliation: Minapharm Pharmaceuticals, Cairo, Egypt. mueller@minapharm.com

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.

Conclusion: The optimized production process described for rSAK-2 in Hansenula polymorpha provides an excellent, economically superior, manufacturing platform for a promising therapeutic fibrinolytic agent.

Show MeSH
Related in: MedlinePlus