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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

Biomass growth over time of a glycerol fed-batch culture and a methanol induced culture, respectively, grown in SYN6 media. The Fed-Batch culture was pulsed twice at t = 1 and 2 with a mixture of 10 g/L glycerol and 10 g/L peptone (final concentration). At four points of time (1–4) a sample was taken. For the other culture, methanol was given once (1) at a final concentration of 10 g/L. Samples were taken at t = 1 and 3. Fermentation was carried out at 1mL scale in the BioLector® system. Samples were analysed in a Western Blot for rSAK-2 expression (5 μL supernatant loaded). Standards (std) are drawn for the individual gels. Final lanes were loaded with THR-174 (~109 ng).
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Figure 5: Biomass growth over time of a glycerol fed-batch culture and a methanol induced culture, respectively, grown in SYN6 media. The Fed-Batch culture was pulsed twice at t = 1 and 2 with a mixture of 10 g/L glycerol and 10 g/L peptone (final concentration). At four points of time (1–4) a sample was taken. For the other culture, methanol was given once (1) at a final concentration of 10 g/L. Samples were taken at t = 1 and 3. Fermentation was carried out at 1mL scale in the BioLector® system. Samples were analysed in a Western Blot for rSAK-2 expression (5 μL supernatant loaded). Standards (std) are drawn for the individual gels. Final lanes were loaded with THR-174 (~109 ng).

Mentions: In further micro-scale experiments using SYN6 medium supplemented with 10 g/L wheat peptone, two feed strategies were assessed in parallel. In the first, a ‘pulsed’ glycerol-peptone fed-batch fermentation was carried out. The feed solution was pulsed manually at the end of the exponential growth phase when the dissolved oxygen (DO) signal increased. This was done twice during fermentation at the time points indicated (Figure 5). At time points 1 to 4, samples were taken. In the second, a culture was induced with methanol at time point 1. The methanol-induced culture received no additional nutrients during fermentation and samples were taken at time points 1 and 3.


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)

Biomass growth over time of a glycerol fed-batch culture and a methanol induced culture, respectively, grown in SYN6 media. The Fed-Batch culture was pulsed twice at t = 1 and 2 with a mixture of 10 g/L glycerol and 10 g/L peptone (final concentration). At four points of time (1–4) a sample was taken. For the other culture, methanol was given once (1) at a final concentration of 10 g/L. Samples were taken at t = 1 and 3. Fermentation was carried out at 1mL scale in the BioLector® system. Samples were analysed in a Western Blot for rSAK-2 expression (5 μL supernatant loaded). Standards (std) are drawn for the individual gels. Final lanes were loaded with THR-174 (~109 ng).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Biomass growth over time of a glycerol fed-batch culture and a methanol induced culture, respectively, grown in SYN6 media. The Fed-Batch culture was pulsed twice at t = 1 and 2 with a mixture of 10 g/L glycerol and 10 g/L peptone (final concentration). At four points of time (1–4) a sample was taken. For the other culture, methanol was given once (1) at a final concentration of 10 g/L. Samples were taken at t = 1 and 3. Fermentation was carried out at 1mL scale in the BioLector® system. Samples were analysed in a Western Blot for rSAK-2 expression (5 μL supernatant loaded). Standards (std) are drawn for the individual gels. Final lanes were loaded with THR-174 (~109 ng).
Mentions: In further micro-scale experiments using SYN6 medium supplemented with 10 g/L wheat peptone, two feed strategies were assessed in parallel. In the first, a ‘pulsed’ glycerol-peptone fed-batch fermentation was carried out. The feed solution was pulsed manually at the end of the exponential growth phase when the dissolved oxygen (DO) signal increased. This was done twice during fermentation at the time points indicated (Figure 5). At time points 1 to 4, samples were taken. In the second, a culture was induced with methanol at time point 1. The methanol-induced culture received no additional nutrients during fermentation and samples were taken at time points 1 and 3.

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