Molecular Parasitology Laboratory, Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran; Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran.
Molecular Parasitology Laboratory, Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran.
J Biotechnol. 2017 Oct 10;259:30-38. doi: 10.1016/j.jbiotec.2017.08.015. Epub 2017 Aug 18.
Human fibroblast growth factor-1 (FGF-1) has powerful mitogenic activities in a variety of cell types and plays significant roles in many physiological processes e.g. angiogenesis and wound healing. There is increasing demand for large scale production of recombinant human FGF-1 (rhFGF-1), in order to investigate the potential medical use. In the present study, we explored SHuffle™ T7 strain for production of rhFGF-1.
A synthetic gene encoding Met-140 amino acid form of human FGF-1 was utilized for expression of the protein in three different E. coli hosts (BL21 (DE3), Rosetta-gami™ 2(DE3), SHuffle™ T7). Total expressions and soluble/insoluble expression ratios of rhFGF-1 in different hosts were analyzed and compared. Soluble rhFGF-1 produced in SHuffle™ T7 cells was purified using one-step heparin-Sepharose affinity chromatography and characterized by a variety of methods for physicochemical and biological properties.
The highest level of rhFGF-1 expression and maximum soluble/insoluble ratio were achieved in SHuffle™ T7 strain. Using a single-step heparin-Sepharose chromatography, about 1500mg of purified rhFGF-1 was obtained from one liter of the culture, representing purification yield of ∼70%. The purified protein was reactive toward anti-FGF-1 ployclonal antibody in immunoblotting. Mass spectrometry confirmed the protein had expected amino acid sequence and molecular weight. In reverse-phase high-performance liquid chromatography (RP-HPLC), the protein displayed the same retention time with the human FGF-1 standard, and purity of 94%. Less than 0.3% of the purified protein was comprised of oligomers and/or aggregates as judged by high-performance size-exclusion chromatography (HP-SEC). Secondary and tertiary structures of the protein, investigated by circular dichroism and intrinsic fluorescence spectroscopy methods, respectively, represented native folding of the protein. The purified rhFGF-1 was bioactive and stimulated proliferation of NIH 3T3 cells with EC50 of 0.84ng/mL.
Although SHuffle™ T7 has been introduced for production of disulfide-bonded proteins in cytoplasm, we herein successfully recruited it for high yield production of soluble and bioactive rhFGF-1, a protein with 3 free cysteine and no disulfide bond. To our knowledge, this is the highest-level of rhFGF-1 expression in E. coli reported so far. Extensive physicochemical and biological analysis showed the protein had similar characteristic to authentic FGF-1.
人成纤维细胞生长因子-1（FGF-1）在多种细胞类型中具有强大的有丝分裂活性，在许多生理过程中发挥着重要作用，例如血管生成和伤口愈合。为了研究其潜在的医学用途，对重组人 FGF-1（rhFGF-1）的大规模生产需求日益增加。在本研究中，我们探索了 SHuffle™ T7 菌株用于 rhFGF-1 的生产。
利用编码人 FGF-1 的 Met-140 个氨基酸形式的合成基因，在三种不同的大肠杆菌宿主（BL21（DE3）、Rosetta-gami™ 2（DE3）、SHuffle™ T7）中表达该蛋白。分析并比较了不同宿主中 rhFGF-1 的总表达量和可溶/不可溶表达比。使用一步肝素琼脂糖亲和层析从 SHuffle™ T7 细胞中纯化可溶性 rhFGF-1，并通过多种方法对其理化和生物学特性进行了表征。
在 SHuffle™ T7 菌株中获得了 rhFGF-1 的最高表达水平和最大可溶性/不可溶性比。使用一步肝素琼脂糖层析，从 1 升培养物中获得了约 1500mg 的纯化 rhFGF-1，代表了约 70%的纯化收率。纯化的蛋白在免疫印迹中对抗 FGF-1 多克隆抗体有反应。质谱分析证实该蛋白具有预期的氨基酸序列和分子量。在反相高效液相色谱（RP-HPLC）中，该蛋白的保留时间与人类 FGF-1 标准品相同，纯度为 94%。高效尺寸排阻色谱（HP-SEC）判断，纯化的蛋白中只有不到 0.3%的蛋白为寡聚物和/或聚集体。通过圆二色性和内源荧光光谱法分别研究了该蛋白的二级和三级结构，表明该蛋白具有天然折叠。纯化的 rhFGF-1 具有生物活性，能刺激 NIH 3T3 细胞增殖，EC50 为 0.84ng/mL。
尽管 SHuffle™ T7 已被引入用于细胞质中环化蛋白的生产，但我们在此成功地招募它用于可溶性和生物活性 rhFGF-1 的高产生产，rhFGF-1 是一种具有 3 个游离半胱氨酸但没有二硫键的蛋白。据我们所知，这是迄今为止大肠杆菌中 rhFGF-1 表达水平最高的一次。广泛的理化和生物学分析表明，该蛋白的特征与天然 FGF-1 相似。
