| Escherichia coli is a popular platform for production of recombinant pharmaceutical proteins,because of its clear genetic background,low cost and rapid proliferation.However,most of recombinant pharmaceutical proteins are expressed as the form of inclusion body,such as recombinant human interleukin 15?rhIL-15?.We took rhIL-15 as the target protein,build up an intein mediated recombinant pharmaceutical proteins soluble expression and purification system in E.coli.rhIL-15 is of great clinical potential for its lymphocyte homeostatic and NK cell development functions.However,the clinical manufacturing of rhIL-15 has been demonstrated to be very challenging due to its expression and purification difficulties.In this study,we used an intein-mediated novel system to produce rhIL-15 in Escherichia coli.We screened several solubilizing tags fused with the self-cleavable Mtu RecA mini intein?I-CM,and demonstrated that Zbasic-?I-CM fusion tag can extraordinarily improve both the solubility and the expressing level of IL-15.The fusion protein‘Zbasic-?I-CM-IL-15'was expressed with high solubility and purified by the cost-effective cation-exchange chromatography.The self-cleavage of tag was then induced and the mature rhIL-15 without N-terminal methionine was released and further purified by hydrophobic interaction and anion-exchange chromatography.The Zbasic-?I-CM fusion tag showed high cleavage activity at pH6/25?,cleavage activation energy was 7.48 kcal/mol.The final product was analyzed by RP-HPLC and UPLC-MS to be of high purity and right mass.The purity was 92%.Molecular weight was 12769.5 Da.The bioactivity was evaluated by a CTLL-2 cell proliferation based assay,and the EC50 was about0.12 ng/mL.By avoiding the time-consuming denaturing-refolding steps in previously reported processes,our method is efficient and cost-effective,and can potentially apply to manufacturing of recombinant pharmaceutical proteins. |
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