Soluble expression of disulfide bond containing proteins FGF15 and FGF19 in the cytoplasm of Escherichia coli.
Soluble expression of disulfide bond containing proteins FGF15 and FGF19 in the cytoplasm of Escherichia coli.
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DOI：
10.1371/journal.pone.0085890
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Guo GL
中科院分区：
文献类型：
--
作者：
Kong B;Guo GL
成纤维细胞生长因子 19 (FGF19) 是小鼠 FGF15 的人类直系同源物，这两种蛋白都充当内分泌信号来调节各种肝功能。 FGF15/FGF19蛋白含有两个二硫键。由于细菌胞质还原环境，不利于大肠杆菌（E. coli）胞质中形成二硫键。细胞质还原环境的修饰和/或蛋白质伴侣的共表达是在大肠杆菌中表达含有二硫键的蛋白质的常见策略。在本研究中，我们报道了一种在大肠杆菌细胞质中生产可溶性 FGF15/FGF19 蛋白的方法。使用几种破坏硫醇-氧化还原途径和/或共表达氧化还原酶或重折叠伴侣的商业菌株来开发这种在大肠杆菌中表达 FGF15/FGF19 的新方法。大肠杆菌中硫醇-二硫键还原途径的突变或单独的硫氧还蛋白 (TRX) N 端融合不足以支持 FGF15/19 蛋白中二硫键的形成。然而，TRX 融合蛋白提高了硫醇-氧化还原系统突变体菌株中 FGF19 的溶解度。此外，单独在硫醇-氧化还原系统突变体中共表达的 DsbC 与 TRX 融合标签的组合改善并进一步增强了 FGF19 的溶解度。可溶性FGF19蛋白可以通过Ni-NTA亲和层析和阴离子交换层析轻松纯化，并且通过抑制小鼠肝脏Cyp7a1基因转录和激活HepG2细胞中的ERK1/2信号通路证实纯化的蛋白保持其生物活性。相反，使用这些策略，细胞质中的可溶性 FGF15 蛋白仍然非常低。总之，我们成功开发了一种在原核细胞中表达功能性FGF19蛋白的方法，并且该策略可能适用于其他含二硫键蛋白的表达。
Fibroblast growth factor 19 (FGF19) is the human ortholog of mouse FGF15, and both proteins function as an endocrine signal to regulate various liver functions. FGF15/FGF19 protein contains two disulfide bonds. It is unfavorable to form disulfide bonds in Escherichia coli (E. coli) cytoplasm because of the bacterial cytoplasmic reducing environment. Modification of the cytoplasmic reducing environment and/or co-expression of protein chaperones are common strategies to express disulfide bond containing proteins in E. coli. In the current study, we report a method to produce soluble FGF15/FGF19 protein in cytoplasm of E. coli. Several commercial available strains with the disruption of thiol-redox pathways, and/or co-expression of redoxase or refolding chaperones were used to develop this novel method for expression of FGF15/FGF19 in E. coli. Mutation of the thiol-disulfide bond reducing pathway in E. coli or N-terminal fusion of thioredox (TRX) alone is not enough to support disulfide bond formation in FGF15/19 proteins. However, TRX fusion protein improved FGF19 solubility in strains of thiol-redox system mutants. In addition, DsbC co-expressed in thiol-redox system mutants alone improved and further enhanced FGF19 solubility with combination of TRX fusion tag. The soluble FGF19 proteins were easily purified through Ni-NTA affinity chromatography and anion exchange chromatography, and the purified protein maintained its biological activities, confirmed by suppressing hepatic Cyp7a1 gene transcription in mice and by activating ERK1/2 signaling pathway in HepG2 cells. In contrast, soluble FGF15 protein in cytoplasm remained very low using these strategies. In summary, we have successfully developed a method to express functional FGF19 protein in prokaryotic cells, and this strategy may be adapted for the expression of other disulfide-containing proteins.
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