A strategy for high-level expression of soluble and functional human interferon α as a GST-fusion protein in E.coli
A strategy for high-level expression of soluble and functional human interferon α as a GST-fusion protein in E.coli
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DOI：
10.1093/protein/gzm012
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发表时间：
2007-05-01
影响因子：
2.4
通讯作者：
Fathallah, Dahmani M.
中科院分区：
文献类型：
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作者：
Rabhi-Essafi, Imen;Sadok, Amine;Fathallah, Dahmani M.
大肠杆菌是生产重组蛋白最广泛使用的宿主。然而，大多数真核蛋白通常是不溶的，错误折叠的包涵体，需要溶解和再折叠。为了在大肠杆菌中实现可溶性重组人干扰素α (rhIFN α)的高水平表达，我们首先构建了重组表达质粒（pGEX-hIFN α 2b），并将hIFN α 2b cDNA与可诱导启动子下游的谷胱甘肽s转移酶（GST）编码序列合并。利用该质粒，我们在优化的培养生长温度和诱导物（IPTG）浓度等环境因素下，利用大肠杆菌BL21菌株，实现了可溶性rhIFN α 2b作为GST融合蛋白70%的表达。然而，凝血酶消化融合蛋白释放IFN片段的效果并不理想。因此，我们设计了表达盒来优化GST-IFN连接处的氨基酸序列，并在凝血酶裂解位点引入大肠杆菌首选密码子。我们使用工程质粒（pGEX-Delta-hIFN α 2b）和修饰的大肠杆菌trxB(-)/gor(-) （Origami）菌株克服了在表达可溶性rhIFNa2b时去除GST片段的问题。我们的结果表明，在没有优化任何步骤的情况下，以100 mg/l的产率生产可溶性和功能性rhIFN α 2b。纯化的可溶性干扰素α 2b与WHO干扰素α标准相比，其比生物活性为2.0 × 10(8) IU/mg。我们的数据首次表明，在大肠杆菌中可以实现高产量生产可溶性和功能性的GST标记的rhIFN α 2b。
Escherichia coli is the most extensively used host for the production of recombinant proteins. However, most of the eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. To achieve high-level expression of soluble recombinant human interferon alpha (rhIFN alpha) in E.coli, we have first constructed a recombinant expression plasmid (pGEX-hIFN alpha 2b), in which we merged the hIFN alpha 2b cDNA with the glutathione S-transferase (GST) coding sequence downstream of the tac-inducible promoter. Using this plasmid, we have achieved 70% expression of soluble rhIFN alpha 2b as a GST fusion protein using E.coli BL21 strain, under optimized environmental factors such as culture growth temperature and inducer (IPTG) concentration. However, release of the IFN moiety from the fusion protein by thrombin digestion was not optimal. Therefore, we have engineered the expression cassette to optimize the amino acid sequence at the GST-IFN junction and to introduce E.coli preferred codon within the thrombin cleavage site. We have used the engineered plasmid (pGEX-Delta-hIFN alpha 2b) and the modified E.coli trxB(-)/gor(-) (Origami) strain to overcome the problem of removing the GST moiety while expressing soluble rhIFNa2b. Our results show the production of soluble and functional rhIFN alpha 2b at a yield of 100 mg/l, without optimization of any step of the process. The specific biological activity of the purified soluble rhIFN alpha 2b was equal to 2.0 x 10(8) IU/mg when compared with the WHO IFN alpha standard. Our data are the first to show that high yield production of soluble and functional rhIFN alpha 2b tagged with GST can be achieved in E.coli.