Interleukin 24 (IL-24) has a broad spectrum of specific antitumor activities without affecting normal cells. The recombinant human IL-24 (rhIL-24) expressed in E. coli has low biological activity due to lack of necessary glycosylation modification. In this study, based on the modification of the non-glycosylated IL-24 with polyethylene glycol (PEG), we aimed to improve the stability and prolong its half-life in vivo. Firstly, the recombinant plasmid containing the hIL-24 cDNA was prepared by the prokaryotic-expression plasmid pET-28a and transformed into E. coli BL21. After induced by isopropyl β-D-thiogalactoside (IPTG), the target protein rhIL-24 was expressed as insoluble inclusion body, which was solubilized and denatured by 6 M guanidine hydrochloride. The denatured rhIL-24 was diluted to refold in the optimized buffer overnight at the protein concentration of 0.1 mg/mL. The refolded rhIL-24 was mainly in the form of soluble aggregate, but high-purity monomer rhIL-24 was obtained through size exchange chromatography with the addition of SDS in elution buffer. The tertiary structure of rhIL-24 was confirmed by fluorescence spectroscopy. Western blot analysis showed that rhIL-24 could be site-specifically modified by mPEG5000-ALD. Methyl thiazolyl tetrazolium (MTT) assay showed no significant difference between mPEG5000-ALD-rhIL-24 and rhIL-24 in inhibiting the growth of melanoma cell line A375 in vitro. Pharmacokinetic studies showed that PEG modification could significantly improve the stability and prolong the half-life of rhIL-24 from 8.41 to 13.2 h. The data strongly suggested that mPEG-ALD 5000 could site-specifically modify rhIL-24 expressed in E. coli. The PEG modification significantly prolonged the half-life of rhIL-24 without reducing its antitumor activity in vitro.

中文翻译：

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重组人白介素24在大肠杆菌中的表达，纯化和聚乙二醇位点修饰。

白介素24（IL-24）具有广泛的特异性抗肿瘤活性，而不会影响正常细胞。由于缺乏必要的糖基化修饰，在大肠杆菌中表达的重组人IL-24（rhIL-24）具有较低的生物学活性。在这项研究中，基于聚乙二醇（PEG）对非糖基化IL-24的修饰，我们旨在提高稳定性并延长其在体内的半衰期。首先，通过原核表达质粒pET-28a制备含有hIL-24 cDNA的重组质粒，并将其转化到大肠杆菌中。BL21。经异丙基β-D-硫代半乳糖苷（IPTG）诱导后，目标蛋白rhIL-24被表达为不溶性包涵体，并被6 M盐酸胍溶解并变性。将变性的rhIL-24稀释，以0.1 mg / mL的蛋白质浓度在优化的缓冲液中重新折叠过夜。重新折叠的rhIL-24主要为可溶性聚集体形式，但通过大小交换色谱法在洗脱缓冲液中添加SDS可获得高纯度单体rhIL-24。rhIL-24的三级结构通过荧光光谱法确认。蛋白质印迹分析显示，rhIL-24可以被mPEG5000-ALD进行位点特异性修饰。甲基噻唑基四唑（MTT）分析显示，mPEG5000-ALD-rhIL-24和rhIL-24在抑制黑素瘤细胞系A375体外生长方面无显着差异。药代动力学研究表明，PEG修饰可以显着改善rhIL-24的稳定性并将其半衰期从8.41小时延长至13.2 h。数据强烈表明mPEG-ALD 5000可以位点特异性修饰rhIL-24大肠杆菌。PEG修饰可显着延长rhIL-24的半衰期，而不会降低其体外抗肿瘤活性。