Efficient Recombinant Production in Mammalian Cells Using a Novel IR/MAR Gene Amplification Method

Yoshio Araki,Chiemi Noguchi,Tetsuro Hamafuji,Noriaki Shimizu,Baochuan Lin

doi:10.1371/journal.pone.0041787

Yoshio Araki, Chiemi Noguchi + Show 3 more

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https://doi.org/10.1371/journal.pone.0041787

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Journal: PloS one	Publication Date: Jul 23, 2012
Citations: 42	License type: CC BY 4.0

Affiliation: Hiroshima University

Abstract

We previously found that plasmids bearing a mammalian replication initiation region (IR) and a nuclear matrix attachment region (MAR) efficiently initiate gene amplification and spontaneously increase their copy numbers in animal cells. In this study, this novel method was applied to the establishment of cells with high recombinant antibody production. The level of recombinant antibody expression was tightly correlated with the efficiency of plasmid amplification and the cytogenetic appearance of the amplified genes, and was strongly dependent on cell type. By using a widely used cell line for industrial protein production, CHO DG44, clones expressing very high levels of antibody were easily obtained. High-producer clones stably expressed the antibody over several months without eliciting changes in both the protein expression level and the cytogenetic appearance of the amplified genes. The integrity and reactivity of the protein produced by this method was fine. In serum-free suspension culture, the specific protein production rate in high-density cultures was 29.4 pg/cell/day. In conclusion, the IR/MAR gene amplification method is a novel and efficient platform for recombinant antibody production in mammalian cells, which rapidly and easily enables the establishment of stable high-producer cell clone.

Highlights

Production of recombinant proteins in cultured mammalian cells is becoming more critical as the need for large amounts of pharmaceuticals protein, e.g. humanized antibody, is increasing rapidly
We previously found that the initiation region (IR)/matrix attachment region (MAR) plasmid could be amplified and used to generate chromosomal homogeneously staining regions (HSR) and/or extrachromosomal double minutes (DMs) of varying size in human colorectal carcinoma COLO 320 cells [6,7]
To investigate IR/MAR plasmid amplification in several Chinese hamster ovary (CHO) cell sublines that are frequently used for antibody production, we transfected the cells with pBM-MycLH (Fig. 1D) coding for the antibody heavy and light chain genes with the IR/MAR sequence or pV-MycLH (Fig. 1E) coding for the antibody heavy and light chain genes without the IR/MAR sequence

Summary

Introduction

Production of recombinant proteins in cultured mammalian cells is becoming more critical as the need for large amounts of pharmaceuticals protein, e.g. humanized antibody, is increasing rapidly. Efficient antibody production by the IR/MAR gene amplification method.

Results

Conclusion