Engineering Research Center of Bioreactor and Pharmaceutical Development, Ministry of Education, Jilin Agricultural University, China.
BMC Biotechnol. 2010 Feb 17;10:14. doi: 10.1186/1472-6750-10-14.
Fibroblast growth factor 21 (FGF21) is a promising drug candidate to combat metabolic diseases. However, high-level expression and purification of recombinant FGF21 (rFGF21) in Escherichia coli (E. coli) is difficult because rFGF21 forms inclusion bodies in the bacteria making it difficult to purify and obtain high concentrations of bioactive rFGF21. To overcome this problem, we fused the FGF21 with SUMO (Small ubiquitin-related modifier) by polymerase chain reaction (PCR), and expressed the fused gene in E. coli BL21(DE3).
By inducing with IPTG, SUMO-FGF21 was expressed at a high level. Its concentration reached 30% of total protein, and exceeded 95% of all soluble proteins. The fused protein was purified by DEAE sepharose FF and Ni-NTA affinity chromatography. Once cleaved by the SUMO protease, the purity of rFGF21 by high performance liquid chromatography (HPLC) was shown to be higher than 96% with low endotoxin level (<1.0 EU/ml). The results of in vivo animal experiments showed that rFGF21 produced by using this method, could decrease the concentration of plasma glucose in diabetic rats by streptozotocin (STZ) injection.
This study demonstrated that SUMO, when fused with FGF21, was able to promote its soluble expression of the latter in E. coli, making it more convenient to purify rFGF21 than previously. This may be a better method to produce rFGF21 for pharmaceutical research and development.
成纤维细胞生长因子 21（FGF21）是一种有前途的药物候选物，可用于治疗代谢疾病。然而，由于重组 FGF21（rFGF21）在大肠杆菌（E. coli）中形成包涵体，使其难以纯化并获得高浓度的生物活性 rFGF21，因此在大肠杆菌中高水平表达和纯化 rFGF21 较为困难。为了解决这个问题，我们通过聚合酶链反应（PCR）将 FGF21 与 SUMO（小泛素相关修饰物）融合，并在大肠杆菌 BL21（DE3）中表达融合基因。
通过 IPTG 诱导，SUMO-FGF21 得到了高水平表达。其浓度达到总蛋白的 30%，超过所有可溶性蛋白的 95%。融合蛋白通过 DEAE 琼脂糖 FF 和 Ni-NTA 亲和层析进行纯化。一旦被 SUMO 蛋白酶切割，通过高效液相色谱（HPLC）纯化的 rFGF21 的纯度超过 96%，内毒素水平低于 1.0 EU/ml。体内动物实验结果表明，用这种方法生产的 rFGF21 可降低链脲佐菌素（STZ）注射诱导的糖尿病大鼠血浆葡萄糖浓度。
本研究表明，SUMO 与 FGF21 融合后，能够促进后者在大肠杆菌中的可溶性表达，使其比以前更方便地纯化 rFGF21。这可能是一种更好的生产 rFGF21 的方法，用于药物研发。
