Transient gene expression: Recombinant protein production with suspension-adapted HEK293-EBNA cells

被引:141
作者
Meissner, P
Pick, H
Kulangara, A
Chatellard, P
Friedrich, K
Wurm, FM [1 ]
机构
[1] Swiss Fed Inst Technol, Dept Chem, Lab Cellular Biotechnol, CH-1015 Lausanne, Switzerland
[2] Swiss Fed Inst Technol, Dept Chem, Lab Phys Chem Polymers & Membranes, CH-1015 Lausanne, Switzerland
[3] Univ Lausanne, Lab Mol Biotechnol, Lausanne, Switzerland
关键词
transient transfection; HEK293-EBNA; calcium phosphate; suspension;
D O I
10.1002/bit.1179
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Transient gene expression (TGE) in mammalian cells at the reactor scale is becoming increasingly important for the rapid production of recombinant proteins. We improved a process for transient calcium phosphate-based transfection of HEK293-EBNA cells in a 1-3 L bioreactor volume. Cells were adapted to suspension culture using a commercially available medium (Bio-Whittaker, Walkersville, MD). Process parameters were optimized using a plasmid reporter vector encoding the enhanced green fluorescent protein (EGFP/CLONTECH, Palo Alto, CA, USA). Using GFP as a marker-protein, we observed by microscopic examination transfection efficiencies between 70-100%. Three different recombinant proteins were synthesized within a timeframe of 7 days from time of transfection to harvest. The first, a human recombinant IgG(1)-type antibody, was secreted into the supernatant of the cell culture and achieved a final concentration of >20 mg/L. An E. coli-derived DNA-binding protein remained intracellular, as expected, but accumulated to such a concentration that the lysate of cells, taken up into the entire culture volume, gave a concentration of 18 mg/L. The third protein, a transmembrane receptor, was expressed at 3-6 x 10(6) molecules/cell. (C) 2001 John Wiley & Sons, Inc.
引用
收藏
页码:197 / 203
页数:7
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