Efficient production of recombinant human FVII in CHO cells using the piggyBac transposon system

IF 1.2 4区生物学 Q4 BIOCHEMICAL RESEARCH METHODS Protein expression and purification Pub Date : 2025-05-01 Epub Date: 2025-01-22 DOI:10.1016/j.pep.2025.106666

Zhen Yang , Xueyun Wang , Shan Luo , Hui Li , Jiangbo Xu , Linlin Liang , Zhimin He , Guangyuan Wang , Zhuobin Wu , Nan Zhong , Haijun Xiang , Zhan Zhang , Caiping Guo , Yunjia Zhang , Fei Yan

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Abstract

As an important coagulation factor, activated coagulation factor VII (FVIIa) is mainly used to treat the bleeding of hemophilia patients who have developed inhibitory antibodies against FVIII and FIX conventional treatment. Recombinant human factor VII (rhFVII) produced in mammalian cell lines have been developed as the most important resource of FVIIa. However, cell lines express rhFVII protein derived from an exogenous expression vector at a lower level than most other proteins. In the current study, we have shown efficient rhFVII production in CHO cell lines using piggyBac (PB) transposon system. rhFVII is successfully expressed in fed-batch culture of CHO cells, and the expression of rhFVII up to 100 mg/L. Moreover, the purified secreted rhFVII was determined by SDS-PAGE and Western Blot. The coagulation activity was determined by the chromogenic Activity ELISA kit. In conclusion, this study has demonstrated that the piggyBac transposon system can be used for an efficient production of recombinant FVII.

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利用piggyBac转座子系统在CHO细胞中高效生产重组人FVII。

活化凝血因子VII （activated coagulation factor VII, FVIIa）作为一种重要的凝血因子，主要用于治疗血友病患者对FVIII产生抑制抗体和FIX常规治疗后的出血。在哺乳动物细胞系中产生的重组人因子VII （rhFVII）已成为重组人因子VII最重要的来源。然而，细胞系表达来自外源表达载体的rhFVII蛋白的水平低于大多数其他蛋白。在目前的研究中，我们已经证明了使用piggyBac （PB）转座子系统在CHO细胞系中有效地产生rhFVII。rhFVII在CHO细胞补料分批培养中成功表达，表达量可达100 mg/L。纯化后的rhFVII通过SDS-PAGE和Western Blot检测。采用显色活性ELISA试剂盒测定凝血活性。总之，本研究表明，piggyBac转座子系统可用于高效生产重组FVII。

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来源期刊

Protein expression and purification 生物-生化研究方法

CiteScore

3.70

自引率

6.20%

发文量

120

审稿时长

32 days

期刊介绍： Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.