Guo J H, Abedi M, Aust S D
Biotechnology Center, Utah State University, Logan 84322-4705, USA.
Arch Biochem Biophys. 1996 Nov 1;335(1):197-204. doi: 10.1006/abbi.1996.0498.
The full-length genes for the heavy (H) and light (L) chains of ferritin isolated from a rat liver cDNA library were amplified using polymerase chain reaction. Each was inserted at the unique BglII site downstream of the p10 promoter of the baculovirus transfer vector pAcUW21. The genes were transferred separately to infectious Autographa californica nuclear polyhedrosis virus (AcNPV) expression vectors after in vivo homologous recombination. Ferritin homopolymers of either H or L chain were expressed up to approximately 1.5 mg per 100 ml of infected cultures (2.0 x 10(6) cells/ml) of Spodoptera frugiperda, Sf-21, 4 days postinfection. Both recombinant H chain ferritin (rH-Ft) and recombinant L chain ferritin (rL-Ft) assembled as multi-subunit complexes with predicted electrophoretic mobility. Neither rH-Ft nor rL-Ft homopolymers had ferroxidase activity in 50 mM NaCl, as we have reported previously for native ferritin [D. DeSilva, D. M. Miller, D.W. Reif, and S.D. Aust (1992) Arch. Biochem. Biophys. 293,409-415]. When ceruloplasmin, a copper-containing protein, was used as a ferroxidase, rH-Ft loaded iron at rates comparable those obtained with native rat liver apoferritin, but rL-Ft failed to load any iron. The initial rate of Fe(II) oxidation catalyzed by ceruloplasmin was increased in the presence of rH-Ft or rat liver ferritin but not in the presence of rL-Ft. A maximum of about 2500 atoms of iron were incorporated into both rH-Ft and rat liver ferritin. These results demonstrate that both rat liver rH-Ft and rL-Ft homopolymer can be properly produced by the baculovirus expression system and ceruloplasmin can only load iron into H chain ferritin. The physiological significance of these results is discussed.
从大鼠肝脏cDNA文库中分离出铁蛋白重链（H）和轻链（L）的全长基因，使用聚合酶链反应进行扩增。将每个基因插入杆状病毒转移载体pAcUW21的p10启动子下游的独特BglII位点。体内同源重组后，将这些基因分别转移到感染性苜蓿银纹夜蛾核型多角体病毒（AcNPV）表达载体中。感染后4天，在每100 ml的草地贪夜蛾Sf-21培养物（2.0×10⁶个细胞/ml）中，H链或L链的铁蛋白同聚物表达量高达约1.5 mg。重组H链铁蛋白（rH-Ft）和重组L链铁蛋白（rL-Ft）均组装成具有预测电泳迁移率的多亚基复合物。正如我们之前对天然铁蛋白的报道[D. DeSilva，D. M. Miller，D. W. Reif，和S. D. Aust（1992年）《生物化学与生物物理学报》293，409 - 415]，在50 mM NaCl中，rH-Ft和rL-Ft同聚物均没有铁氧化酶活性。当使用含铜蛋白铜蓝蛋白作为铁氧化酶时，rH-Ft加载铁的速率与天然大鼠肝脏脱铁铁蛋白相当，但rL-Ft未能加载任何铁。在rH-Ft或大鼠肝脏铁蛋白存在的情况下，铜蓝蛋白催化的Fe（II）氧化初始速率增加，但在rL-Ft存在的情况下没有增加。rH-Ft和大鼠肝脏铁蛋白中最多可掺入约2500个铁原子。这些结果表明，杆状病毒表达系统可以正确产生大鼠肝脏rH-Ft和rL-Ft同聚物，并且铜蓝蛋白只能将铁加载到H链铁蛋白中。讨论了这些结果的生理学意义。
