Chen Xin, Hu Yueyue, Xu Hongyi, Wang Xiaoyan, Deng Kai
Reproductive Medicine Center, Renmin Hospital, Hubei University of Medicine, Shiyan 442000, Hubei, China.
School of Basic Medical Sciences, Hubei University of Medicine, Shiyan 442000, Hubei, China.
Sheng Wu Gong Cheng Xue Bao. 2019 Jun 25;35(6):1135-1142. doi: 10.13345/j.cjb.180507.
PLCζ is a new isoenzyme of the PLC family which plays an important role in activating mammalian oocytes. In recent years, large-scale expression and purification of active PLCζ protein in vitro for structural biology research has not been successful. In this study, the recombinant human PLCζ protein was expressed and purified in the baculovirus expression system. First, the full length of human PLCζ gene was cloned into the pFastBac-HTA plasmid to form the recombinant donor plasmid that was further transformed into DH10Bac Escherichia coli cells to construct the recombined bacmid by the site-specific transposition that was screened by resistance and blue-white spots. Then the bacmid was transfected to Sf9 insect cells via cellfectin to package the recombinant baculovirus. After the amplification of the recombinant baculovirous, the recombinant protein was expressed from the cells transduced by the recombinant baculovirus and was purified by Ni-NTA resin. Purified protein was identified by Western blotting and time-of-flight mass spectrometry and the enzyme activity was determined. The results showed that the recombinant PLCζ protein in the Sf9 cells was achieved at 72 hours after baculovirus infection and expressed in secreted form in cell culture medium. The recombinant protein purified by Ni²⁺ affinity column was identified as PLCζ by Western blotting and ionization time-of-flight mass spectrometry and the enzyme activity was up to 326.8 U/mL. The experimental results provide a reference for the large-scale production and biological application of recombinant human PLCζ protein.
磷脂酶Cζ（PLCζ）是磷脂酶C（PLC）家族的一种新的同工酶，在激活哺乳动物卵母细胞中起着重要作用。近年来，为进行结构生物学研究在体外大规模表达和纯化活性PLCζ蛋白尚未成功。在本研究中，重组人PLCζ蛋白在杆状病毒表达系统中进行表达和纯化。首先，将人PLCζ基因全长克隆到pFastBac-HTA质粒中，形成重组供体质粒，进一步将其转化到DH10Bac大肠杆菌细胞中，通过抗性和蓝白斑筛选的位点特异性转座构建重组杆粒。然后通过细胞转染试剂将杆粒转染到Sf9昆虫细胞中包装重组杆状病毒。重组杆状病毒扩增后，由重组杆状病毒转导的细胞表达重组蛋白，并通过镍-亚氨基二乙酸（Ni-NTA）树脂进行纯化。通过蛋白质免疫印迹法（Western blotting）和飞行时间质谱对纯化的蛋白进行鉴定，并测定酶活性。结果显示，杆状病毒感染后72小时在Sf9细胞中获得重组PLCζ蛋白，且以分泌形式表达于细胞培养基中。经镍离子亲和柱纯化的重组蛋白通过蛋白质免疫印迹法和电离飞行时间质谱鉴定为PLCζ，酶活性高达326.8 U/mL。实验结果为重组人PLCζ蛋白的大规模生产和生物学应用提供了参考。
