Baldi Lucia, Muller Natalie, Picasso Sylviane, Jacquet Raymond, Girard Philippe, Thanh Huy Phan, Derow Elisabeth, Wurm Florian M
Laboratory of Cellular Biotechnology, Institute for Biological Engineering and Biotechhnology, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
Biotechnol Prog. 2005 Jan-Feb;21(1):148-53. doi: 10.1021/bp049830x.
Recent advances in genomics, proteomics, and structural biology raised the general need for significant amounts of pure recombinant protein (r-protein). Because of the difficulty in obtaining in some cases proper protein folding in bacteria, several methods have been established to obtain large amounts of r-proteins by transgene expression in mammalian cells. We have developed three nonviral DNA transfer protocols for suspension-adapted HEK-293 and CHO cells: (1) a calcium phosphate based method (Ca-Pi), (2) a calcium-mediated method called Calfection, and (3) a polyethylenimine-based method (PEI). The first two methods have already been scaled up to 14 L and 100 L for HEK-293 cells in bioreactors. The third method, entirely serum-free, has been successfully applied to both suspension-adapted CHO and HEK-293 cells. We describe here the application of this technology to the transient expression in suspension cultivated HEK-293 EBNA cells of some out of more than 20 secreted r-proteins, including antibodies, dimeric proteins, and tagged proteins of various complexity. Most of the proteins were expressed from different plasmid vectors within 5-10 days after the availability of the DNA. Transfections were successfully performed from the small scale (1 mL in 12-well microtiter plates) to the 2 L scale. The results reported made it possible to establish an optimized cell culture and transfection protocol that minimizes batch-to-batch variations in protein expression. The work presented here proves the applicability and robustness of transient transfection technology for the expression of a variety of recombinant proteins.
基因组学、蛋白质组学和结构生物学的最新进展引发了对大量纯重组蛋白（r蛋白）的普遍需求。由于在某些情况下难以在细菌中实现适当的蛋白质折叠，已建立了几种通过在哺乳动物细胞中进行转基因表达来获得大量r蛋白的方法。我们已经为适应悬浮培养的HEK-293和CHO细胞开发了三种非病毒DNA转染方案：（1）基于磷酸钙的方法（Ca-Pi），（2）一种称为Calfection的钙介导方法，以及（3）基于聚乙烯亚胺的方法（PEI）。前两种方法已在生物反应器中扩大规模至14 L和100 L用于HEK-293细胞。第三种方法完全无血清，已成功应用于适应悬浮培养的CHO和HEK-293细胞。我们在此描述了该技术在悬浮培养的HEK-293 EBNA细胞中对20多种分泌型r蛋白中的一些进行瞬时表达的应用，这些r蛋白包括抗体、二聚体蛋白以及各种复杂程度的标签蛋白。大多数蛋白在DNA可用后的5 - 10天内从不同的质粒载体表达。转染成功地从小规模（12孔微量滴定板中1 mL）扩大到2 L规模。所报道的结果使得能够建立一种优化的细胞培养和转染方案，将蛋白质表达中的批次间差异降至最低。本文所展示的工作证明了瞬时转染技术对于多种重组蛋白表达的适用性和稳健性。
