Simple and Efficient Establishment of Recombinant Protein Hyper-Producing Cells by Using RAS-Amplified CHO Cell Line

Abstract

Chinese hamster ovary (CHO) cells is a widely used host cell line and can mass-produce recombinant proteins by using various amplifiable selectable marker gene and selection drugs. However, the establishment of highly productive cell line is a laborious and time-consuming work. In the present study, we developed a novel efficient method. We transfected CHO cells with plasmid expressing a gene of interest along with that expressing cell surface marker protein, and we selected recombinant CHO cells by their expression of cell surface marker protein and seed them into 96-well plates by single cell sorting. This method enabled a rapid and efficient establishment of recombinant CHO cells without drug selection. Furthermore, by using ras-amplified CHO cell line (CHO-hp1) as host cells, recombinant protein hyper-producing CHO cells can be easily obtained within a relatively short period. Actually, larger number of recombinant clones were obtained by using CHO-hp1 cells as host cells (188 clones/1,000 seeded wells), while clones cannot be obtained by using conventional CHO cells as host cells (0 clones/1,000 seeded wells). Furthermore, these recombinant cells obtained from CHO-hp1 cells demonstrated higher productivity (45 mg/106 cells/day). These results demonstrate that this method enables a rapid and efficient establishment of recombinant protein hyper-producing cell lines, and can be applicable to production of various recombinant proteins.