Molecular Biotechnology. Bernard R. Glick

      Some selection schemes are designed not only to identify transfected cells, but also to increase heterologous-protein production by amplifying the copy number of the expression vector. The dihydrofolate reductase–methotrexate system falls into this category. Dihydrofolate reductase catalyzes the reduction of dihydrofolate to tetrahydrofolate, which is required for the production of purines. The enzyme is inhibited by methotrexate. Sensitivity to methotrexate can be overcome if the cell produces excess dihydrofolate reductase, and as the methotrexate concentration is increased over time, the dihydrofolate reductase gene in cultured cells is amplified. It is not unusual for methotrexate-resistant cells to have hundreds of dihydrofolate reductase genes. The standard dihydrofolate reductase–methotrexate protocol entails transfecting dihydrofolate reductase-deficient cells with an expression vector carrying a dihydrofolate reductase gene as the selectable marker gene and treating the cells with methotrexate. After the initial selection of transfected cells, the concentration of methotrexate is gradually increased, and eventually cells with very high copy numbers of the expression vector are selected.

Engineering Mammalian Cell Hosts for Enhanced Productivity

      In large-scale bioreactors, depleted nutrients and accumulation of toxic cell waste can limit the viability and density of cells as they respond to stress by inducing cell death, also known as apoptosis. One method to improve cell growth and viability under culture conditions in bioreactors is to prevent the tumor suppressor protein p53, which is a transcription factor, from activating the cell death response pathway. The mouse double mutant 2 protein (MDM2) binds to protein p53 and prevents it from acting as a transcription factor (Fig. 3.43). MDM2 also marks p53 for degradation. CHO cells were transfected with plasmids containing a regulatable MDM2 gene and cultured under conditions that mimicked the late stages of cell culture and in nutrient-limited medium. Cultures expressing MDM2 had higher cell densities and delayed cell death compared to nontransfected cells, especially in nutrient-deprived medium.

      Figure 3.43 Strategy to increase yields of recombinant mammalian cells. Cell death (apoptosis), stimulated by the transcription factor p53, can lead to decreased yields of recombinant mammalian cells grown under stressful conditions in large bioreactors. To prevent cell death, the gene encoding MDM2 is introduced into mammalian cells. The MDM2 protein binds to p53 and prevents it from inducing expression of proteins required for apoptosis. Engineered cells not only showed delayed cell death, but also achieved higher cell densities in bioreactors.

      Many cultured mammalian cells are unable to achieve high cell densities in cultures because toxic metabolic products accumulate in the culture medium and inhibit cell growth. Although efforts are made to optimize the culture conditions, inevitably nutrients essential for optimal cell growth, including oxygen, are reduced. Under low-oxygen conditions, many cells, including CHO cells, secrete the acidic waste product lactate as they struggle to obtain energy from glucose. Under these conditions, pyruvate, an intermediate compound produced during the metabolism of glucose, is converted to lactate by lactate dehydrogenase rather than entering into the tricarboxylic acid cycle, where it is further oxidized through the activity of pyruvate carboxylase (Fig. 3.44). To counteract the acidification of the medium from lactate secretion, the human pyruvate carboxylase gene was cloned into an expression vector under the control of the cytomegalovirus (CMV) promoter and the SV40 polyadenylation signals and transfected into CHO cells. When the pyruvate carboxylase gene was stably integrated into the CHO genome and expressed, the enzyme was detected in the mitochondria, where glucose is degraded. After 7 days in culture, the rate of lactate production decreased by up to 40% in the engineered cells.

      Figure 3.44 When oxygen is present, pyruvate, which is formed from glucose during glycolysis, is converted by the enzyme pyruvate carboxylase to an intermediate compound in the tricarboxylic acid (TCA) cycle. This metabolic pathway is important for the generation of cellular energy and for the synthesis of biomolecules required for cell proliferation. However, under low-oxygen conditions, such as those found in large bioreactors, pyruvate carboxylase has a low level of activity. Under these conditions, lactate dehydrogenase converts pyruvate into lactate, which yields a lower level of energy. Cultured cells secrete lactate, thereby acidifying the medium.

      Many of the eukaryotic DNA viruses from which the vectors used in mammalian cells are derived maintain their genomes as multicopy episomal DNA (plasmids) in the host cell nucleus. These viruses produce proteins, such as the large-T antigen in SV40 and the nuclear antigen 1 protein in Epstein-Barr virus, that help to maintain the plasmids in the host nucleus and to ensure that each host cell produced after cell division receives a copy of the plasmid. To increase the copy number of the target gene by increasing the plasmid copy number, cell lines have been engineered to express the SV40 large-T antigen or Epstein-Barr nuclear antigen 1.

      Many proteins of therapeutic value, such as antibodies and interferon, are secreted. However, the high levels of these proteins that are desirable from a commercial standpoint can quickly overwhelm the capacity of the cell secretory system. Thus, protein processing is a major limiting step in the achievement of high target protein yields. Although high levels of recombinant protein production have been found to increase the levels of proteins associated with proper protein folding and secretion in the endoplasmic reticulum, the levels are usually not sufficient for optimal protein processing. Researchers have therefore devised methods to increase the capacity for protein secretion by engineering cell lines with enhanced production of components of the secretion apparatus. In this regard, an effective strategy may be to simultaneously overexpress several, if not all, of the proteins that make up the secretory mechanism. This can be achieved through the enhanced production of the transcription factor X box protein 1 (Xbp-1), a key regulator of the secretory pathway. Normally, full-length, unspliced xbp-1 mRNA is found in nonstressed cells and is not translated into a stable, functional protein (Fig. 3.45A). However, when unfolded or misfolded proteins accumulate in the endoplasmic reticulum, a ribonuclease is activated that specifically cleaves xbp-1 mRNA (Fig. 3.45B). This results in the production of a functional transcription factor that activates the expression of a number of proteins of the secretion apparatus. A truncated xbp-1 gene that encodes an actively translated form of xbp-1 mRNA (Fig. 3.45C) was overexpressed under the control of the CMV promoter in recombinant CHO cell lines that were previously constructed to express human erythropoietin, human γ-interferon, and human monoclonal antibodies either stably or transiently. Expression of the genes encoding proteins of the secretion apparatus that are controlled by Xbp-1 was found to increase in response to elevated levels of Xbp-1. Although overexpression of Xbp-1 did not increase the production of recombinant proteins in stable cell lines in which the target gene is inserted in a chromosome, a significant increase was observed in cell lines engineered to express the target proteins transiently from a plasmid-encoded gene.

      Figure 3.45 Strategy to increase yields of secreted recombinant proteins from mammalian cells by simultaneously upregulating the expression of several proteins in the secretion apparatus. The expression of chaperones and other proteins of the secretion apparatus is controlled by the transcription factor Xbp-1. (A) In unstressed cells, the intron (green box) is not cleaved from the xbp-1 transcript, and therefore, functional Xbp-1 transcription factor is not produced. (B) However, in stressed cells that have accumulated misfolded proteins, an endoribonuclease