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Highly efficient soluble expression and purification of recombinant human basic fibroblast growth factor (hbFGF) by fusion with a new collagen-like protein (Scl2) in Escherichia coli.
Preparative Biochemistry & Biotechnology ( IF 2.0 ) Pub Date : 2020-02-06 , DOI: 10.1080/10826068.2020.1721533
Inamur Rahman 1, 2, 3 , Lina Fang 4 , Zhang Wei 4 , Xiaodong Zheng 1 , Lian Jiazhang 2, 3 , Lei Huang 2, 3 , Zhinan Xu 2, 3
Affiliation  

Human basic fibroblast growth factor (hbFGF) is involved in a wide range of biological activities that affect the growth, differentiation, and migration. Due to its wound healing effects and therapy, hbFGF has the potential as therapeutic agent. Therefore, large-scale production of biologically active recombinant hbFGF with low cost is highly desirable. However, the complex structure of hbFGF hinders its high-level expression as the soluble and functional form. In the present study, an efficient, cost-effective, and scalable method for producing recombinant hbFGF was developed. The modified collagen-like protein (Scl2-M) from Streptococcus pyogenes was used as the fusion tag for producing recombinant hbFGF for the first time. After optimization, the expression level of Scl2-M-hbFGF reached approximately 0.85 g/L in the shake flask and 7.7 g/L in a high cell-density fermenter using glycerol as a carbon source. Then, the recombinant Scl2-M-hbFGF was readily purified using one-step acid precipitation and the purified Scl2-M-hbFGF was digested with enterokinase. The digested mixture was further subject to ion-exchange chromatography, and the final high-purity (96%) hbFGF product was prepared by freeze-drying. The recovery rate of the whole purification process attained 55.0%. In addition, the biological activity of recombinant hbFGF was confirmed by using L929 and BALB/c3T3 fibroblasts. Overall, this method has the potential for large scale production of recombinant hbFGF.



中文翻译:

重组人碱性成纤维细胞生长因子(hbFGF)与新型胶原样蛋白(Scl2)融合后在大肠杆菌中的高效可溶性表达和纯化。

人类碱性成纤维细胞生长因子(hbFGF)参与影响生长,分化和迁移的多种生物学活动。由于其伤口愈合作用和治疗作用,hbFGF具有作为治疗剂的潜力。因此,非常需要以低成本大规模生产生物活性重组hbFGF。但是,hbFGF的复杂结构阻碍了其作为可溶性和功能性形式的高水平表达。在本研究中,开发了一种高效,经济,可扩展的重组hbFGF生产方法。化脓性链球菌的修饰胶原样蛋白(Scl2-M)首次用作重组hbFGF的融合标签。优化后,摇瓶中Scl2-M-hbFGF的表达水平达到约0.85 g / L,使用甘油作为碳源的高细胞密度发酵罐中的表达水平达到7.7 g / L。然后,使用一步酸沉淀法即可轻松纯化重组Scl2-M-hbFGF,并用肠激酶消化纯化的Scl2-M-hbFGF。将消化后的混合物进一步进行离子交换层析,并通过冷冻干燥制备最终的高纯度(96%)hbFGF产物。整个纯化过程的回收率达到55.0%。另外,通过使用L929和BALB / c3T3成纤维细胞证实了重组hbFGF的生物学活性。总体而言,该方法具有大规模生产重组hbFGF的潜力。

更新日期:2020-02-06
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