Liu Chaoting, Dalby Brian, Chen Weixing, Kilzer Jennifer M, Chiou Henry C
Research and Development, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008, USA.
Mol Biotechnol. 2008 Jun;39(2):141-53. doi: 10.1007/s12033-008-9051-x.
The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.
将克隆基因高效转染到哺乳动物细胞系统中，对于大量生产重组蛋白（r蛋白）起着关键作用。为了建立一个简单且可扩展的瞬时蛋白生产系统，我们使用了一种基于阳离子脂质的转染试剂——FreeStyle MAX，来研究在无血清悬浮培养的人胚肾（HEK）293细胞和中国仓鼠卵巢（CHO）细胞中的瞬时转染。我们通过定量绿色荧光蛋白（GFP）来监测转染效率，并通过检测克隆的人IgG抗体的表达来监测r蛋白的产生。对转染试剂浓度、DNA浓度、复合物形成时间以及转染时的细胞密度等参数进行了分析和优化。在最佳条件下，HEK-293细胞和CHO细胞中均获得了约70%的GFP阳性细胞以及50 - 80 mg/l的分泌型IgG抗体。将转染系统扩大到1升规模后，转染效率和蛋白产量相似。此外，我们还评估了在HEK-293细胞和CHO细胞中生产治疗性蛋白，如人促红细胞生成素和人凝血因子IX的情况。我们的结果表明，在无血清适应的悬浮培养哺乳动物细胞中，使用最佳的瞬时转染条件可获得更高产量的蛋白。
