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一种让胰岛素前体生产翻倍的方法

2010-5-28  阅读(2642)

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德国亥姆霍兹中心感染研究所25日发表公报说,该所发现了能令胰岛素前体产量翻倍的方法,并决定不注册而公开所有技术细节,以使更多的、尤其是发展中国家的糖尿病患者能够得到药物治疗。

目前,临床治疗中使用的胰岛素制剂是由转化胰岛素前体制成的,胰岛素前体主要在大肠杆菌或酿酒酵母菌中生产。该所在此基础上发现了一种新方法,能令胰岛素前体的产量翻倍,从而降低胰岛素生产的成本。这一方法生产的胰岛素经证实能够安全地在人体应用。

公报说,糖尿病已经不再是以往认为的“富贵病”。发展中国家的患者人数增长迅速,但许多贫穷国家患者因买不起药而饱受折磨甚至失去生命,其重要原因是许多新药和方法申请了保护,导致治疗费用高昂。亥姆霍兹中心感染研究所这一研究项目的目标就是降低胰岛素生产成本,让更多糖尿病患者受益。

上海劲马生物()推荐原文出处:

Microbial Cell Factories  doi:10.1186/1475-2859-9-31

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

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 alpha-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 c*tion 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.

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