Stabilization of Protein Expression in Mammalian Cells Employing a Toxin/Antitoxin Based Strategy

Abstract

Recombinant protein expression in mammalian cells is a key tool for research and industrial application. Introduction of transgenes into cells is usually accompanied with random integration of the expression cassette into the genomic DNA of the host. Their expression level strongly depends on the integration site. Due to epigenetic effects expression of the recombinant gene frequently drops over time. These low-expressing cells compete with the transgene over-expressors, often leading to overgrowth, even in presence of antibiotic selection. Here we describe a new method by which transgene expression is stabilized in extensively proliferating cultures even in cases where the transgene opposes mild disadvantages to the expressing cells. The method is based on strict co-expression of the transgene with an anti-toxin in cells that express the respective toxin. Since the strength of anti-toxin expression correlates with an advantage for cell growth, the cells with strong anti-toxin expression succeed over time in cultures of heterogeneous cells. The application of this principle was exemplified in CHO cells. We show that high transgene expression can be achieved in cells with a tightly controlled toxin expression by a simple coupling of the antitoxin to the transgene of interest. Importantly, production is stable even in absence of selectable drugs. Together, it represents a novel approach for development of high producing cells.