Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae.
Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae.
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DOI：
10.1002/bit.24409
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发表时间：
2012-05
影响因子：
3.8
通讯作者：
Nielsen, Jens
中科院分区：
文献类型：
--
作者：
Liu, Zihe;Tyo, Keith E. J.;Martinez, Jose L.;Petranovic, Dina;Nielsen, Jens
关键词：
酿酒酵母已成为生产商品和特种化学品和蛋白质（如工业酶和药用蛋白质）的有吸引力的细胞工厂。在这里，我们评估了重组蛋白分泌的最重要的表达因子：我们选择了两种不同的蛋白质（胰岛素前体（IP）和α-淀粉酶），两种不同的表达载体（POTud质粒和CPOTud质粒）和两种前导序列（糖基化的α因子前导序列和没有糖基化位点的合成前导序列）。我们使用IP和α-淀粉酶分别作为简单蛋白和多结构域蛋白以及非糖基化蛋白和糖基化蛋白的代表。将编码两种重组蛋白的基因与两种不同的前导序列独立融合，并使用两种不同的质粒系统表达，得到8种不同的菌株，通过分批发酵进行评价。在所有表达系统中，IP的分泌水平（μmol/L）均高于α-淀粉酶，且不同载体的IP产量差异较大。我们还发现，在二次转换过程中蛋白质生产动力学发生了变化，即在葡萄糖摄取阶段以更高的速率生产IP，而在乙醇摄取阶段以更高的速率生产淀粉酶。为了比较，我们还参考了来自另一项研究的数据，其中我们使用了p426 GPD质粒（使用URA 3作为标记基因和pGPD 1作为表达启动子的标准载体，（Tyo KEJ，et al. Submitted））。对于IP，与标准的基于URA 3的载体相比，CPOTud载体的蛋白质产量高出10倍以上，因此该载体系统代表了用于酵母中重组蛋白质生产的未来研究和优化的宝贵资源。
Yeast Saccharomyces cerevisiae has become an attractive cell factory for production of commodity and speciality chemicals and proteins, such as industrial enzymes and pharmaceutical proteins. Here we evaluate most important expression factors for recombinant protein secretion: we chose two different proteins (insulin precursor (IP) and α-amylase), two different expression vectors (POTud plasmid and CPOTud plasmid) and two kinds of leader sequences (the glycosylated alpha factor leader and a synthetic leader with no glycosylation sites). We used IP and α-amylase as representatives of a simple protein and a multi-domain protein, as well as a non-glycosylated protein and a glycosylated protein, respectively. The genes coding for the two recombinant proteins were fused independently with two different leader sequences and were expressed using two different plasmid systems, resulting in eight different strains that were evaluated by batch fermentations. The secretion level (μmol/L) of IP was found to be higher than that of α-amylase for all expression systems and we also found larger variation in IP production for the different vectors. We also found that there is a change in protein production kinetics during the diauxic shift, i.e. the IP was produced at higher rate during the glucose uptake phase, whereas amylase was produced at a higher rate in the ethanol uptake phase. For comparison, we also refer to data from another study, in which we used the p426GPD plasmid (standard vector using URA3 as marker gene and pGPD1 as expression promoter, (Tyo KEJ, et al. Submitted)). For the IP there is more than 10 fold higher protein production with the CPOTud vector compared with the standard URA3-based vector, and this vector system therefore represent a valuable resource for future studies and optimization of recombinant protein production in yeast.
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