Engineering Research Center of Bioreactor and Pharmaceutical Development, Ministry of Education, Jilin Agricultural University, Changchun, 130118, China.
Appl Microbiol Biotechnol. 2014 Jan;98(2):695-704. doi: 10.1007/s00253-013-4929-3. Epub 2013 Apr 30.
Using fusion tags, expression of recombinant human fibroblast growth factor 18 (rhFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, including chondrogenesis and osteogenesis, hair growth, and neuroprotection. However, high-level rhFGF18 expression is difficult and the products are often not homogeneous. Furthermore, fusion-tagged protein has higher immunogenicity and lower bioactivity, and the removal of the fused tag is expensive. To overcome the limitations of fusion-tagged expression of protein and to prepare soluble highly bioactive rhFGF18, we have developed a rapid and efficient expression strategy. Optimized hFGF18 gene was amplified by polymerase chain reaction and cloned into pET22b and pET3c vectors, then transformed into E. coli strains Origima (DE3) and BL21 (DE3)PlysS. The best combination of plasmid and host strain was selected, and only Origima (DE3)/pET3c-rhFGF18 was screened for high-level expressed rhFGF18. Under optimal conditions in a 30-L fermentor, the average bacterial yield and expression level of rhFGF18 of three batches were more than 652 g and 30 % respectively, after treatment with 1 mM isopropyl-thio-β-galactopyranoside for 10 h at 25 °C. The target protein was purified by CM Sepharose FF and heparin affinity chromatography. The purity of rhFGF18 was shown by HPLC to be higher than 95 %, and the yield was 155 mg/L. In vitro MTT assays demonstrated that the purified rhFGF18 could stimulate significant proliferation of NIH3T3 cells, and animal experiments showed that rhFGF18 could effectively regulate hair growth. In conclusion, this may be a better method of producing rhFGF18 to meet the increasing demand in its pharmacological application.
利用融合标签，已广泛应用于哺乳动物细胞和大肠杆菌中表达重组人成纤维细胞生长因子 18（rhFGF18），用于基础研究和临床应用，包括软骨形成和骨形成、毛发生长和神经保护。然而，高水平的 rhFGF18 表达是困难的，且产物通常不均匀。此外，融合标签蛋白具有更高的免疫原性和更低的生物活性，并且去除融合标签是昂贵的。为了克服融合标签表达蛋白的局限性，并制备可溶性的高生物活性 rhFGF18，我们开发了一种快速有效的表达策略。通过聚合酶链反应扩增优化的 hFGF18 基因，并将其克隆到 pET22b 和 pET3c 载体中，然后转化到 Origima（DE3）和 BL21（DE3）PlysS 大肠杆菌菌株中。选择了质粒和宿主菌株的最佳组合，仅筛选 Origima（DE3）/pET3c-rhFGF18 用于高水平表达 rhFGF18。在 30-L 发酵罐中优化条件下，三批 rhFGF18 的平均细菌产量和表达水平分别超过 652 g 和 30%，在 25°C 下用 1mM异丙基-β-D-硫代半乳糖苷处理 10 h 后。目标蛋白通过 CM Sepharose FF 和肝素亲和层析进行纯化。HPLC 显示 rhFGF18 的纯度高于 95%，收率为 155 mg/L。体外 MTT 测定表明，纯化的 rhFGF18 能显著刺激 NIH3T3 细胞增殖，动物实验表明 rhFGF18 能有效调节毛发生长。总之，这可能是一种更好的生产 rhFGF18 的方法，以满足其在药理学应用中的日益增长的需求。
