Comparative analysis of different biofactories for the production of a major diabetes autoantigen
Comparative analysis of different biofactories for the production of a major diabetes autoantigen
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DOI：
10.1007/s11248-013-9749-9
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发表时间：
2014-04-01
影响因子：
3
通讯作者：
Pezzotti, Mario
中科院分区：
文献类型：
--
作者：
Avesani, Linda;Merlin, Matilde;Pezzotti, Mario
人谷氨酸脱羧酶（hGAD65）的65-kDa异构体是一种主要的糖尿病自身抗原，可用于自身免疫性糖尿病的诊断和（最近）治疗。我们之前报道了一种催化无活性版本（hGAD65mut）在转基因烟草植物中积累的水平比其活性对应物高10倍，与哺乳动物生产平台相比，提供了一种安全且更便宜的蛋白质来源。在这里，我们发现hGAD65mut在烟叶（使用pK7WG2或MagnICON载体）、杆状病毒载体在昆虫细胞中的瞬时表达，以及使用诱导表达系统在细菌细胞中的瞬时表达也比hGAD65产生更高的水平，尽管后者系统不适合，因为hGAD65mut在内含体中积累。这些平台中产量最高的是MagnICON系统，其产量达到78.8 μ g/g鲜叶重（FLW），但这远远低于表现最好的精英转基因烟草植株，经过6代自交后，其产量达到114.3 μ g/g FLW。尽管育种过程耗时3年，但该转基因系统被认为是最具生产力和成本效益的。MagnICON系统总体上产量较低，但在几天内产生了大量蛋白质。因此，这两种基于植物的系统都比我们手中基于杆状病毒的生产平台有优势。
The 65-kDa isoform of human glutamic acid decarboxylase (hGAD65) is a major diabetes autoantigen that can be used for the diagnosis and (more recently) the treatment of autoimmune diabetes. We previously reported that a catalytically-inactive version (hGAD65mut) accumulated to tenfold higher levels than its active counterpart in transgenic tobacco plants, providing a safe and less expensive source of the protein compared to mammalian production platforms. Here we show that hGAD65mut is also produced at higher levels than hGAD65 by transient expression in Nicotiana benthamiana (using either the pK7WG2 or MagnICON vectors), in insect cells using baculovirus vectors, and in bacterial cells using an inducible-expression system, although the latter system is unsuitable because hGAD65mut accumulates within inclusion bodies. The most productive of these platforms was the MagnICON system, which achieved yields of 78.8 mu g/g fresh leaf weight (FLW) but this was substantially less than the best-performing elite transgenic tobacco plants, which reached 114.3 mu g/g FLW after six generations of self-crossing. The transgenic system was found to be the most productive and cost-effective although the breeding process took 3 years to complete. The MagnICON system was less productive overall, but generated large amounts of protein in a few days. Both plant-based systems were therefore advantageous over the baculovirus-based production platform in our hands.