Suárez-Patiño Sandra Fernanda, Mancini Renato Astray, Pereira Carlos Augusto, Suazo Claudio Alberto Torres, Mendonça Ronaldo Zucatelli, Jorge Soraia Attie Calil
Laboratório de Imunologia Viral, Instituto Butantan, São Paulo, SP, Brazil; Departamento de Engenharia Química, Universidade Federal de São Carlos, São Carlos, SP, Brazil.
Laboratório de Imunologia Viral, Instituto Butantan, São Paulo, SP, Brazil.
J Biotechnol. 2014 Dec 20;192 Pt A:255-62. doi: 10.1016/j.jbiotec.2014.05.027. Epub 2014 Jul 7.
The transient transfection process has been developed to allow rapid production of recombinant proteins. In this paper, we describe the transient expression of recombinant rabies virus glycoprotein (RVGP) in Drosophila melanogaster Schneider 2 (S2) cells. Different cell transfection reagents were evaluated, together with the effects of different cell cultivation procedures on RVGP expression. Yields of RVGP in the range 50-90ng/10(7) cells were obtained in multi-well plate transfection experiments, where it was observed that RVGP expression was linked to the DNA concentration. RVGP expression was 1.3 times higher using 10μg rather than 5μg of DNA. Inhibition of RVGP expression was observed at higher concentrations of DNA, with DNA concentrations above 15μg decreasing RVGP expression 1.5-fold for cells transfected with polyethylenimine (PEI) and 1.6-fold for cells transfected with cationic lipid. The results of shake flask transfection indicated that S2 cells were more effectively transfected in suspension than under static conditions. RVGP yields of 182.2ng/10(7) cells (PEI), 201ng/10(7) cells (calcium phosphate), and 215ng/10(7) cells (cationic lipid) were obtained for S2 cell suspension cultures. The highest volumetric RVGP concentration (309ng/mL) was found for cells transfected with cationic lipid. This value was 1.21 and 1.16 times higher, respectively, than for cells transfected with PEI (253.4ng/mL) and calcium phosphate (237.2ng/mL). There was little effect of transfection on the kinetics of cell growth, with growth rates of 1.12 and 1.19d(-1) for transfected and control cells, respectively. In spinner flasks, the expression of RVGP was 150 and 138ng/10(7) cells for transfection using PEI and calcium phosphate, respectively. A comparison of the different transfection reagents (calcium phosphate, cationic lipid, and cationic polymer) showed no significant differences in RVGP expression when shake flasks were used. Overall, the data indicated that transient expression in D. melanogaster S2 cells is a practical way of synthesizing RVGP for use in structural and functional studies.
已开发出瞬时转染方法以实现重组蛋白的快速生产。在本文中，我们描述了重组狂犬病病毒糖蛋白（RVGP）在黑腹果蝇Schneider 2（S2）细胞中的瞬时表达。评估了不同的细胞转染试剂，以及不同细胞培养程序对RVGP表达的影响。在多孔板转染实验中获得了50 - 90ng/10⁷细胞范围内的RVGP产量，观察到RVGP表达与DNA浓度相关。使用10μg DNA时RVGP表达比5μg DNA时高1.3倍。在较高DNA浓度下观察到RVGP表达受到抑制，对于用聚乙烯亚胺（PEI）转染的细胞，DNA浓度高于15μg时RVGP表达降低1.5倍，对于用阳离子脂质转染的细胞，DNA浓度高于15μg时RVGP表达降低1.6倍。摇瓶转染结果表明，S2细胞在悬浮状态下比在静态条件下更有效地被转染。对于S2细胞悬浮培养，分别获得了182.2ng/10⁷细胞（PEI）、201ng/10⁷细胞（磷酸钙）和215ng/10⁷细胞（阳离子脂质）的RVGP产量。用阳离子脂质转染的细胞中发现最高的RVGP体积浓度（309ng/mL）。该值分别比用PEI转染的细胞（253.4ng/mL）和磷酸钙转染的细胞（237.2ng/mL）高1.21倍和1.16倍。转染对细胞生长动力学影响很小，转染细胞和对照细胞的生长速率分别为1.12和1.19d⁻¹。在转瓶中，使用PEI和磷酸钙转染时，RVGP的表达分别为150和138ng/10⁷细胞。比较不同的转染试剂（磷酸钙、阳离子脂质和阳离子聚合物）发现，使用摇瓶时RVGP表达没有显著差异。总体而言，数据表明在黑腹果蝇S2细胞中的瞬时表达是合成用于结构和功能研究的RVGP的一种实用方法。
