Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media.
Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media.
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DOI：
10.1186/s12934-017-0721-x
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发表时间：
2017-06-15
影响因子：
6.4
通讯作者：
Ruddock LW
中科院分区：
文献类型：
--
作者：
Gąciarz A;Khatri NK;Velez-Suberbie ML;Saaranen MJ;Uchida Y;Keshavarz-Moore E;Ruddock LW
在大肠杆菌中生产含有二硫键的重组蛋白具有挑战性。在大多数情况下，目的蛋白需要靶向氧化周质或以包涵体的形式在细胞质中表达，然后溶解并在体外重新折叠。这两种方法都有局限性。以前我们发现，可溶性表达的二硫键蛋白质在细胞质中的E。用称为CyDisCo的系统在摇瓶规模上培养大肠杆菌是可能的，该系统基于感兴趣的蛋白质与巯基氧化酶和二硫键异构酶的共表达沿着。使用CyDisCo，可以在细胞质中存在完整还原途径的情况下产生二硫键结合的蛋白质。在这里，我们扩大生产四个二硫键蛋白搅拌罐生物反应器，并实现了高细胞密度和蛋白质产量的葡萄糖补料分批发酵，使用E。coli菌株（BW 25113）的细胞质还原途径完整。即使不进行工艺优化，纯化的人单链抗体IgA 1片段产量也达到139 mg/L，鸡抗生物素蛋白产量达到71 mg/L，而人生长激素1和白细胞介素6的纯化产量约为1 g/L。初步结果表明，在W3110菌株的发酵中，当用甘油代替葡萄糖作为碳源时，人生长激素1也被有效地产生。我们的研究结果首次表明，在大肠杆菌细胞质中高效生产高产量的可溶性二硫键蛋白。还原途径完整的大肠杆菌在化学成分确定的基本培养基上扩大到生物反应器培养是可行的。本文的在线版本（doi：10.1186/s12934-017-0721-x）包含补充材料，可供授权用户使用。
The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm. Here we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA1 antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source. Our results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media. The online version of this article (doi:10.1186/s12934-017-0721-x) contains supplementary material, which is available to authorized users.