Ahmadi Maryam, Mahboudi Fereidoun, Akbari Eidgahi Mohammad Reza, Nasr Reza, Nematpour Fatemeh, Ahmadi Samira, Ebadat Saeedeh, Aghaeepoor Mojtaba, Davami Fatemeh
Medical Biotechnology Dept., Semnan University of Medical Sciences, Semnan, Iran.
Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Biotechnol Prog. 2016 Nov;32(6):1570-1576. doi: 10.1002/btpr.2362. Epub 2016 Sep 30.
Traditional methods to generate CHO cell lines rely on random integration(s) of the gene of interest and result in unpredictable and unstable protein expression. In comparison, site-specific recombination methods increase the recombinant protein expression by inserting transgene at a locus with specific expression features. PhiC31 serine integrase, catalyze unidirectional integration that occurs at higher frequency in comparison with the reversible integration carried out by recombinases such as Cre. In this study, using different ratios of phiC31 serine integrase, we evaluated the phiC31 mediated gene integration for expression of a humanized IgG1 antibody (mAb0014) in CHO-S cells. Light chain (LC) and heavy chain (HC) genes were expressed in one operon under EF1α promoter and linked by internal ribosome entry site (IRES) element. The clonal selection was carried out by limiting dilution. Targeted integration approach increased recombinant protein yield and stability in cell pools. The productivity of targeted cell pools was about 4 mg/L and about 40 µg/L in the control cell pool. The number of integrated transgenes was about 19 fold higher than the control cells pools. Our results confirmed that the phiC31 integrase leads to mAb expression in more than 90% of colonies. The productivity of the PhiC31 integrated cell pools was stable for three months in the absence of selection as compared with conventional transfection methods. Hence, utilizing PhiC31 integrase can increase protein titer and decrease the required time for protein expression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1570-1576, 2016.
传统的生成CHO细胞系的方法依赖于目的基因的随机整合，会导致不可预测且不稳定的蛋白质表达。相比之下，位点特异性重组方法通过将转基因插入具有特定表达特征的位点来提高重组蛋白的表达。PhiC31丝氨酸整合酶催化单向整合，与Cre等重组酶进行的可逆整合相比，其发生频率更高。在本研究中，我们使用不同比例的phiC31丝氨酸整合酶，评估了phiC31介导的基因整合在CHO-S细胞中表达人源化IgG1抗体（mAb0014）的情况。轻链（LC）和重链（HC）基因在EF1α启动子下的一个操纵子中表达，并通过内部核糖体进入位点（IRES）元件连接。通过有限稀释进行克隆选择。靶向整合方法提高了细胞池中的重组蛋白产量和稳定性。靶向细胞池的生产力约为4 mg/L，而对照细胞池约为40 µg/L。整合的转基因数量比对照细胞池高约19倍。我们的结果证实，phiC31整合酶可使90%以上的菌落表达mAb。与传统转染方法相比，PhiC31整合细胞池在无选择的情况下生产力可稳定三个月。因此，利用PhiC31整合酶可提高蛋白滴度并减少蛋白质表达所需的时间。© 2016美国化学工程师学会生物技术进展，32:1570 - 1576，2016。
