Abstract

High-level production of recombinant human interleukin-24 (IL-24), a multifunctional immunomodulatory cytokine, has been challenging due primarily to its aggregation as inclusion bodies in the bacterial host while persistent poor-expression in the insect/mammalian expression systems. The present study presents a robust, vector-host combination (pE-SUMO-IL24), auto-inducible medium (YNG/M9NG), and a simple purification scheme for soluble, bioactive, and cost-effective production of native-like IL-24 (nIL-24) in Escherichia coli. The final protein yield, following a three-step purification scheme (IMAC, SEC, dialysis), was 98 mg/L in shake-flask culture (with scale-up potential), which was several folds higher than reported earlier. In vitro cytotoxicity assays with HeLa and HCT116 cancer cell lines (performed using different concentrations of nIL-24) and the fluorescence activated cell sorting analysis (FACS) revealed a dose- and concentration-dependent increase in the population of pro-apoptotic cells with concomitant, statistically significant drop in the number of cells existent at G_o/G1-, S-, and G2/M-phases (P < 0.002). The bioactive nIL-24, developed through this study, holds promise for use in further functional characterizations/applications.

Key points

• Yeast SUMO fusion partner at N-terminus for improved solubility of an otherwise insoluble IL-24 in E. coli.

• Enhanced cell densities with concomitant several-fold increase in protein yield by lactose-inducible media.

• Improved inhibition of cervical and colorectal carcinomas by native-like nIL-24 compared with Met-containing IL.

• Heterologous nIL-24 may enable better understanding of the functional intricacies linked up with its unique cancer-specific features.

中文翻译：

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基于SUMO融合和自动诱导的组合方法可增强大肠杆菌中生物活性人白介素24的产生

摘要

大规模生产重组人白介素24（IL-24），一种多功能的免疫调节细胞因子，具有挑战性，这主要是由于其作为细菌宿主中的包涵体聚集而在昆虫/哺乳动物表达系统中持续表达不佳。本研究提出了一种强大的载体-宿主组合（p​​E-SUMO-IL24），自动诱导培养基（YNG / M9NG）和一种简单的纯化方案，用于可溶性，生物活性和成本有效的天然类IL-的生产大肠杆菌中的24（nIL-24）。经过三步纯化方案（IMAC，SEC，透析），最终的蛋白质产量在摇瓶培养中（具有扩大规模的潜力）为98 mg / L，比先前报道的高出几倍。使用HeLa和HCT116癌细胞系（使用不同浓度的nIL-24进行的体外细胞毒性试验）和荧光激活细胞分选分析（FACS）显示，促凋亡细胞群中剂量和浓度依赖性增加，并伴随，在G _o / G1-，S-和G2 / M期存在的细胞数量具有统计学意义的下降（P <0.002）。通过这项研究开发的具有生物活性的nIL-24有望用于进一步的功能表征/应用。

关键点

•酵母SUMO N末端的融合伴侣，可改善原本不溶的IL-24在大肠杆菌中的溶解度。

•乳糖诱导型培养基可提高细胞密度，同时蛋白质产量增加数倍。

•与含Met的IL相比，天然的nIL-24对子宫颈癌和结肠直肠癌的抑制作用得到改善。

•异源nIL-24可能有助于更好地了解与其独特的癌症特异性特征相关的功能复杂性。