Human granulocyte colony-stimulating factor (hG-CSF) is a cytokine that regulates the proliferation, maturation, and differentiation of precursor cells to neutrophils. In the present study, we report the feasibility of inducing recombinant hG-CSF expression (rhG-CSF) in a pET vector system by combinatorial induction using low-concentration ethanol, IPTG, and lactose and auto-induction media (AIM). The coding sequence of hG-CSF transcript variant 2 was expressed in pET14 vector, and the effect of combinatorial induction was analyzed on inclusion body (IB) formation, biomass, protein purification, and bioactivity. Results showed that there was an inverse relationship between the temperature and soluble expression of rhG-CSF. Three-step washing with Triton-X, 2 M, and 5 M urea resulted in the maximum recovery of IBs. Combinatorial single-spike induction with IPTG, ethanol, and lactose in a batch culture led to a 3-fold increase in the expression of rhG-CSF. It was also observed that low concentration of ethanol (1-3% v/v) could be used in lieu of IPTG for inducing the rhG-CSF protein expression without adversely affecting biomass production. A 2.4-fold increase in productivity was obtained in LB-AIM media with combinatorial ethanol induction, and the overall yield of 2.8 g/L rhG-CSF was found. The purified rhG-CSF was bioactive and increased the cellular proliferation of umbilical cord blood-derived mesenchymal stem cells (U-MSC) by 29%. In conclusion, our study shows that combined ethanol induction can enhance the expression of rhG-CSF with three-step washing for recovery of the proteins from IBs and a single-step purification of rhG-CSF by affinity chromatography. KEY POINTS: • Low concentration of ethanol (1-3%) could be used in lieu of IPTG for inducing rhG-CSF expression. • Combinatorial single-spike induction with IPTG, ethanol, and lactose improved rhG-CSF expression. • Purified rhG-CSF was bioactive and increased the proliferation of U-MSC.

中文翻译：

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一项对比研究表明，组合乙醇处理可提高重组hG-CSF在大肠杆菌中的整体生产率。

人粒细胞集落刺激因子（hG-CSF）是一种调节前体细胞向中性粒细胞增殖，成熟和分化的细胞因子。在本研究中，我们报告了通过使用低浓度乙醇，IPTG，乳糖和自动诱导培养基（AIM）的组合诱导在pET载体系统中诱导重组hG-CSF表达（rhG-CSF）的可行性。在pET14载体中表达了hG-CSF转录变体2的编码序列，并分析了组合诱导对包涵体（IB）形成，生物量，蛋白质纯化和生物活性的影响。结果表明，温度与rhG-CSF的可溶性表达呈反比关系。用Triton-X，2 M和5 M尿素进行三步洗涤可最大程度地回收IB。在分批培养中，用IPTG，乙醇和乳糖进行组合单穗诱导可导致rhG-CSF表达增加3倍。还观察到可以使用低浓度的乙醇（1-3％v / v）代替IPTG来诱导rhG-CSF蛋白表达，而不会不利地影响生物量的产生。在组合乙醇诱导下，LB-AIM培养基的生产率提高了2.4倍，总产量为2.8 g / L rhG-CSF。纯化的rhG-CSF具有生物活性，可使脐带血来源的间充质干细胞（U-MSC）的细胞增殖增加29％。结论，我们的研究表明，联合乙醇诱导可以通过三步洗涤从IBs中回收蛋白质并通过亲和色谱法一步纯化rhG-CSF来增强rhG-CSF的表达。要点：•可以使用低浓度的乙醇（1-3％）代替IPTG来诱导rhG-CSF表达。•IPTG，乙醇和乳糖的组合单穗诱导可改善rhG-CSF表达。•纯化的rhG-CSF具有生物活性，可增加U-MSC的增殖。