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

Microbial Cell Factories

Research

Ursula Rinas¹  Ahmad Adnan²  Thomas Gäbel³  Chandrasekhar Gurramkonda⁴  Sathyamangalam Swaminathan⁵  Navin Khanna⁵  Dipti Chugh⁵  Natasa Skoko⁶  Sulena Polez⁶  Sergio Tisminetzky⁶ 

[1] Helmholtz Centre for Infection Research, Braunschweig, Germany;Helmholtz Centre for Infection Research, Braunschweig, Germany;Department of Chemistry, Government College University, Lahore, akistan;Helmholtz Centre for Infection Research, Braunschweig, Germany;Fraunhofer ITEM, Hannover/Braunschweig, Germany;Helmholtz Centre for Infection Research, Braunschweig, Germany;International Centre for Genetic Engineering & Biotechnology, New Delhi, India;International Centre for Genetic Engineering & Biotechnology, New Delhi, India;International Centre for Genetic Engineering & Biotechnology, Trieste, Italy;

关键词: Culture Broth;    Human Insulin;    Methanol Concentration;    Immobilize Metal Affinity Chromatography;    Immobilize Metal Affinity Chromatography;   

DOI  :  10.1186/1475-2859-9-31

 received in 2010-01-27, accepted in 2010-05-12,  发布年份 2010

来源: Springer

【 摘 要 】

BackgroundThe 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.ResultsA 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.ConclusionsA 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.

【 授权许可】

Unknown   
© Gurramkonda et al; licensee BioMed Central Ltd. 2010. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.