Efficient production of recombinant parathyroid hormone (rPTH) fragment 1-34 in the methylotrophic yeast Hansenula polymorpha

Abstract

Background: Osteoporosis is a bone disease of the elderly that leads to increased risk of fracture. Currently, it aff ects more than 200 million adults worldwide, placing an enormous economic burden on healthcare providers. Present approved drug treatments of osteoporosis, for example, using bisphosphonates, only reduce bone mineral loss, are variable in prevention of future fractures and can induce serious complications long term. From 2002, availability of the recombinant active, N-terminal 1-34 fragment of human parathyroid hormone 1-34, which directly stimulates bone formation, has provided an alternative, potentially more eff ective therapeutic agent for treating osteoporosis, especially in its most severe form. We have adopted the methylotrophic yeast strain Hansenula polymorpha (H. polymorpha) for production of recombinant parathyroid hormone (rPTH) 1-34 since its expression system is highly inducible and target proteins are secreted intact into the fermentation broth without the need for enzymatic cleavage of a fusion protein. Methods: Culture conditions for the growth of selected clones of H. polymorpha super-transformants and their rPTH production capacity were sequentially optimized through the microtitre plate, shake fl ask, small tank bioreactor scales to the large bioreactor scale. Results: The production of rPTH 1-34 by transformed H. polymorpha was greatly enhanced by adding the calnexin chaperone gene to the expression plasmid. Implementation of fi nal optimized culture medium constituents and operational fermentation conditions, of which maintenance of constant oxygen tension (pO 2) was found to be a critical factor at the 80 litre scale, resulted in yields up to 150 mg active rPTH 1-34 per liter of culture supernatant. Conclusion: A robust fermentation process for the effi cient production of active rPTH 1-34 based on a calnexin super-transformed H. polymorpha clone was developed. Potentially, this has cost-saving benefi ts for the production and marketing of clinical-grade rPTH 1-34.

A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active β-d-Galactosidase from Paracoccus sp. 32d

“…Several endogenous and heterologous signal sequences have also been used to secrete proteins in yeast expression hosts [ 6 , 20 , 22 , 23 ]. Due to the fact that the conservation among eukaryotic signal sequences is strong, the pre-pro-sequence of the S. cerevisiae α-mating factor (α-MF) is commonly used for recombinant proteins secretion in non- Saccharomyces yeasts, e.g., K. lactis [ 6 , 22 ], B. adeninivorans [ 55 ], O. polymorpha [ 55 , 64 , 65 ], and K. phaffii [ 6 , 20 , 66 , 67 , 68 ]. Moreover, the signal sequence usually comprises a Kex2 protease recognition site, which is located between the signal peptide and the heterologous protein for proteolytic maturation.…”

Advances in Using Hansenula polymorpha as Chassis for Recombinant Protein Production

“…Further examples of recombinant proteins using H. polymorpha as host include the human Parathyroid Hormone (PTH) and Staphylokinase (SAK). Here it is summarized how the recombinant strain was constructed, the process was optimized and scaled-up to 80 liters (Moussa et al, 2012; Mueller et al, 2013). The PTH is a hormone secreted by the parathyroid gland responsible for calcium homeostasis in the blood and osteogenesis.…”

“…For that, the partial oxygen pressure (pO2) was set to 30%. The feeding strategy was changed from pulse-wise to feeding scheduled realized at a constant rate ranging from 20 to 60 g/L/h for 52 h. Using this fermentation strategy was obtained 120 mg/L and 150 mg/L PTH in 8 and 80 L, respectively (Mueller et al, 2013).…”

Downstream process development strategies for effective bioprocesses: Trends, progress, and combinatorial approaches

“…This HT cultivation device can be found widely in the literature, e.g. in product formation studies for Corynebacterium glutamicum and Hansenula polymorpha . Scalability of the results in miniature scale can be ensured by the sulfite oxidation method guaranteeing a constant oxygen transfer rate between two scales .…”

“…This HT cultivation device can be found widely in the literature, e.g. in product formation studies for Corynebacterium glutamicum [70,71] and Hansenula polymorpha [72]. Scalability of the www.els-journal.com…”