Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin

verfasst von

Chandrasekhar Gurramkonda, Sulena Polez, Natasa Skoko, Ahmad Adnan, Thomas Gäbel, Dipti Chugh, Sathyamangalam Swaminathan, Navin Khanna, Sergio Tisminetzky, Ursula Rinas

Abstract

Background: The prevalence of diabetes is predicted to rise significantly in the coming decades. A recent analysis projects that by the year 2030 there will be ~366 million diabetics around the world, leading to an increased demand for inexpensive insulin to make this life-saving drug also affordable for resource poor countries.Results: A synthetic insulin precursor (IP)-encoding gene, codon-optimized for expression in P. pastoris, was cloned in frame with the Saccharomyces cerevisiae α-factor secretory signal and integrated into the genome of P. pastoris strain X-33. The strain was grown to high-cell density in a batch procedure using a defined medium with low salt and high glycerol concentrations. Following batch growth, production of IP was carried out at methanol concentrations of 2 g L^-1, which were kept constant throughout the remaining production phase. This robust feeding strategy led to the secretion of ~3 gram IP per liter of culture broth (corresponding to almost 4 gram IP per liter of cell-free culture supernatant). Using immobilized metal ion affinity chromatography (IMAC) as a novel approach for IP purification, 95% of the secreted product was recovered with a purity of 96% from the clarified culture supernatant. Finally, the purified IP was trypsin digested, transpeptidated, deprotected and further purified leading to ~1.5 g of 99% pure recombinant human insulin per liter of culture broth.Conclusions: A simple two-phase cultivation process composed of a glycerol batch and a constant methanol fed-batch phase recently developed for the intracellular production of the Hepatitis B surface antigen was adapted to secretory IP production. Compared to the highest previously reported value, this approach resulted in an ~2 fold enhancement of IP production using Pichia based expression systems, thus significantly increasing the efficiency of insulin manufacture.

Organisationseinheit(en)

Institut für Technische Chemie

Externe Organisation(en)

Helmholtz-Zentrum für Infektionsforschung GmbH (HZI)
International Centre for Genetic Engineering and Biotechnology
Fraunhofer-Institut für Toxikologie und Experimentelle Medizin (ITEM)

Typ

Artikel

Journal

Microbial cell factories

Band

9

ISSN

1475-2859

Publikationsdatum

12.05.2010

Publikationsstatus

Veröffentlicht

Peer-reviewed

Ja

ASJC Scopus Sachgebiete

Biotechnologie, Bioengineering, Angewandte Mikrobiologie und Biotechnologie

Ziele für nachhaltige Entwicklung

SDG 3 – Gute Gesundheit und Wohlergehen

Elektronische Version(en)

https://doi.org/10.1186/1475-2859-9-31 (Zugang: Offen)
http://hdl.handle.net/10033/139961 (Zugang: Offen)