Rabhi-Essafi Imen, Sadok Amine, Khalaf Noureddine, Fathallah Dahmani M
Molecular Biotechnology Group, Institute Pasteur, Tunis, Tunisia.
Protein Eng Des Sel. 2007 May;20(5):201-9. doi: 10.1093/protein/gzm012. Epub 2007 Apr 12.
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 (rhIFNalpha) in E. coli, we have first constructed a recombinant expression plasmid (pGEX-hIFNalpha2b), in which we merged the hIFNalpha2b 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 rhIFNalpha2b 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-hIFNalpha2b) and the modified E. coli trxB(-)/gor(-) (Origami) strain to overcome the problem of removing the GST moiety while expressing soluble rhIFNalpha2b. Our results show the production of soluble and functional rhIFNalpha2b at a yield of 100 mg/l, without optimization of any step of the process. The specific biological activity of the purified soluble rhIFNalpha2b was equal to 2.0 x 10(8) IU/mg when compared with the WHO IFNalpha standard. Our data are the first to show that high yield production of soluble and functional rhIFNalpha2b tagged with GST can be achieved in E. coli.
大肠杆菌是生产重组蛋白时使用最为广泛的宿主。然而，大多数真核蛋白通常以不溶性、错误折叠的包涵体形式获得，需要进行溶解和重折叠。为了在大肠杆菌中实现可溶性重组人干扰素α（rhIFNα）的高水平表达，我们首先构建了一个重组表达质粒（pGEX-hIFNα2b），其中我们将hIFNα2b cDNA与谷胱甘肽S-转移酶（GST）编码序列在tac诱导型启动子下游进行了融合。使用该质粒，在优化的环境因素如培养生长温度和诱导剂（IPTG）浓度下，我们利用大肠杆菌BL21菌株实现了作为GST融合蛋白的可溶性rhIFNα2b的70%表达。然而，通过凝血酶消化从融合蛋白中释放IFN部分并不理想。因此，我们对表达盒进行了工程改造，以优化GST-IFN连接处的氨基酸序列，并在凝血酶切割位点引入大肠杆菌偏好密码子。我们使用了工程化质粒（pGEX-Δ-hIFNα2b）和改造后的大肠杆菌trxB(-)/gor(-)（Origami）菌株，以克服在表达可溶性rhIFNα2b时去除GST部分的问题。我们的结果表明，在未对该过程的任何步骤进行优化的情况下，以100 mg/l的产量生产出了可溶性且有功能的rhIFNα2b。与世界卫生组织IFNα标准相比，纯化后的可溶性rhIFNα2b的比生物活性等于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可溶性且有功能的带GST标签的rhIFNα2b。
