Fusion of the Dhfr/Mtx and IR/MAR gene amplification methods produces a rapid and efficient method for stable recombinant protein production

• Published in: PloS one 2012-12, Vol.7 (12), p.e52990
• Main Authors: Noguchi, Chiemi, Araki, Yoshio, Miki, Daisuke, Shimizu, Noriaki
• Format: Article
• Language: English
• Subjects: Amplification; Analysis; Animals; Biology; Cell culture; CHO Cells; Chromosomes; Cricetinae; Dihydrofolate reductase; DNA methylation; Engineering; Fc receptors; Gene Amplification; Genes; Genetic research; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Methods; Methotrexate; Nucleic Acid Amplification Techniques - methods; Plasmids; Proteins; Receptors, Fc - genetics; Receptors, Fc - metabolism; Recombinant proteins; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Reductase; Replication initiation; Tetrahydrofolate dehydrogenase
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0052990

Amplification of the dihydrofolate reductase gene (Dhfr) by methotrexate (Mtx) exposure is commonly used for recombinant protein expression in Chinese hamster ovary (CHO) cells. However, this method is both time- and labor-intensive, and the high-producing cells that are generated are frequently unstable in culture. Another gene amplification method is based on using a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR), which result in the spontaneous initiation of gene amplification in transfected cells. The IR/MAR and Dhfr/Mtx methods of gene amplification are based on entirely different principles. In this study, we combine these two methods to yield a novel method, termed the IR/MAR-Dhfr fusion method, which was used to express three proteins, the Fc receptor, GFP, and recombinant antibody. The fusion method resulted in a dramatic increase in expression of all three proteins in two CHO sub-lines, DXB-11, and DG44. The IR/MAR-Dhfr fusion amplified the genes rapidly and efficiently, and produced larger amounts of antibody than the Dhfr/Mtx or IR/MAR methods alone. While the amplified structure produced by the Dhfr/Mtx method was highly unstable, and the antibody production rate rapidly decreased with the culture time of the cells, the IR/MAR-Dhfr fusion method resulted in stable amplification and generated clonal cells that produced large amounts of antibody protein over a long period of time. In summary, the novel IR/MAR-Dhfr fusion method enables isolation of stable cells that produce larger amounts of a target recombinant protein more rapidly and easily than either the Dhfr/Mtx or IR/MAR methods alone.