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德國(guó)亥姆霍茲中心感染研究所25日發(fā)表公報(bào)說，該所發(fā)現(xiàn)了能令胰島素前體產(chǎn)量翻倍的方法，并決定不注冊(cè)而公開所有技術(shù)細(xì)節(jié)，以使更多的、尤其是發(fā)展中國(guó)家的糖尿病患者能夠得到藥物治療。
目前，臨床治療中使用的胰島素制劑是由轉(zhuǎn)化胰島素前體制成的，胰島素前體主要在大腸桿菌或釀酒酵母菌中生產(chǎn)。該所在此基礎(chǔ)上發(fā)現(xiàn)了一種新方法，能令胰島素前體的產(chǎn)量翻倍，從而降低胰島素生產(chǎn)的成本。這一方法生產(chǎn)的胰島素經(jīng)證實(shí)能夠安全地在人體應(yīng)用。
公報(bào)說，糖尿病已經(jīng)不再是以往認(rèn)為的“富貴病”。發(fā)展中國(guó)家的患者人數(shù)增長(zhǎng)迅速，但許多貧窮國(guó)家患者因買不起藥而飽受折磨甚至失去生命，其重要原因是許多新藥和方法申請(qǐng)了保護(hù)，導(dǎo)致治療費(fèi)用高昂。亥姆霍茲中心感染研究所這一研究項(xiàng)目的目標(biāo)就是降低胰島素生產(chǎn)成本，讓更多糖尿病患者受益。
上海勁馬生物（）推薦原文出處：
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.
