Xu Dongsheng, Wan Aini, Peng Lin, Chen Yun, He Yang, Yang Jianfeng, Jin Jian
a Laboratory of Molecular Pharmacology, School of Pharmaceutical Sciences , Jiangnan University , Wuxi , China.
b Jiangsu Institute of Hematology , The First Affiliated Hospital of Soochow University , Suzhou , China.
Prep Biochem Biotechnol. 2017 May 28;47(5):489-495. doi: 10.1080/10826068.2016.1275010. Epub 2017 Jan 3.
Hepatocyte growth factor (HGF) is a potent multifunctional cytokine that affects proliferation, migration, and morphogenesis of various cells. HGF is secreted as an inactive single-chain precursor protein and activated by the cleavage of serine proteases to form heterodimers. In our current study, the cleavage site of HGF was blocked by replaced Arg 494 of Glu (R494E) that resulted in the single-chain HGF (R494E) unable to be cleaved by serine proteases. We established Chinese hamster ovary (CHO) cells overexpressing HGF (R494E), the expression of HGF (R494E) achieved 12 mg/L and was similar to a previously reported study. The recombinant protein was then purified from culture medium using a two-step chromatographic procedure that resulted in about a 40% recovery rate. The purified HGF (R494E) was obtained as a single-chain active protein. It concluded that HGF (R494E) exhibited a biologically active protein and the overexpressing CHO cell line supplied sufficient material for future studies. The R494E replacement of the cleavage site would be beneficial to the utility of other similar therapeutic proteins.
肝细胞生长因子（HGF）是一种强大的多功能细胞因子，可影响各种细胞的增殖、迁移和形态发生。HGF以无活性的单链前体蛋白形式分泌，并通过丝氨酸蛋白酶的切割而激活，形成异二聚体。在我们目前的研究中，HGF的切割位点被将Glu的Arg 494替换为（R494E）所阻断，导致单链HGF（R494E）无法被丝氨酸蛋白酶切割。我们建立了过表达HGF（R494E）的中国仓鼠卵巢（CHO）细胞系，HGF（R494E）的表达量达到12mg/L，与先前报道的研究相似。然后使用两步色谱法从培养基中纯化重组蛋白，回收率约为40%。纯化后的HGF（R494E）以单链活性蛋白形式获得。结果表明，HGF（R494E）表现出生物活性蛋白，过表达的CHO细胞系为未来的研究提供了充足的材料。切割位点的R494E替换将有利于其他类似治疗性蛋白质的应用。
