Premaster Production of Protein From Mammalian Cell Culture PDF 2024

Genetic engineering of Animal cells in culture “Mammalian Cell Expression Systems” (Part II) Dr. Dina Amr 1 The core steps involved in producing the desired recombinant protein are similar across the various expression systems...

Genetic engineering of Animal cells in culture “Mammalian Cell Expression Systems” (Part II) Dr. Dina Amr 1 The core steps involved in producing the desired recombinant protein are similar across the various expression systems 2 Mammalian cell expression systems – HEK293 and CHO 3  How to select and amplify the genes of transfected cells “Selectable Markers for Mammalian Expression Vectors” I. Cell selection by genetic markers The proportion of cells that accept exogenous DNA during a transfection process may be as low as 1 in 106 and therefore a means of selection of the transfected cells is essential. To allow selection, a marker gene can be incorporated into a recipient cell. The purpose of the genetic marker is to identify the cells that have been transfected. The marker can be linked to the same DNA vector as the target gene. In fact, a number of bacterial marker genes have been adapted for eukaryotic cells. ‣ For example, the bacterial neo gene, which encodes neomycin phosphotransferase, is often used to select transfected mammalian cells. 4  How to select and amplify the genes of transfected cells “Selectable Markers for Mammalian Expression Vectors” I. Cell selection by genetic markers Types of Genetic Marker genes: a)Non dominant marker genes. b) Dominant marker genes. A. Non dominant marker genes: can only be used for selection in a population of cells that are mutants, lacking the marker enzyme activity prior to transfection. Examples: Gene markers that have been used extensively in mammalian cells include genes for the enzymes hypoxanthine guanine phosphoribosyl transferase (HGPRT), thymidine kinase (TK) and dihydrofolate reductase (DHFR). 5  How to select and amplify the genes of transfected cells “Selectable Markers for Mammalian Expression Vectors” I. Cell selection by genetic markers B. Dominant marker genes: are more useful because they do not require mutant recipient cells and can be used with any cell type. A. Examples: the gene for the enzyme xanthine-guanine phosphoribosyl transferase (XGPRT) which is only present in bacteria and permits the metabolism of xanthine. Only recipient mammalian cells that have acquired the bacterial gene during transfection can survive in a medium containing xanthine, hypoxanthine and mycophenolic acid, which inhibits the conversion of IMP to XMP. B. Antibiotic resistance genes are also suitable as dominant markers. For example, the neomycin-resistance gene (neoR) codes for the enzyme amino-glycoside phosphotransferase. The presence of this enzyme confers resistance to the aminoglycoside group of antibiotics of which G418 is the one most commonly used. G418 prevents replication of mammalian cells. If the neoR gene is expressed then the cells survive. 6  How to select and amplify the genes of transfected cells “Selectable Markers for Mammalian Expression Vectors” II. Gene amplification Some selection schemes are designed not only to identify transfected cells, but to increase heterologous-protein production by amplifying the copy number of the expression vector. This involves an increase in the number of copies of a specific gene per cell and can result in increased synthesis of the corresponding protein. The gene amplification is stable and results in corresponding increases in the enzyme levels in the cell. By this method stable cell lines can be produced with as much as a 1000- fold increase in gene copy number and corresponding protein expression. ‣ For example, The dihydrofolate reductase–methotrexate system falls into this category. 7  How to select and amplify the genes of transfected cells “Selectable Markers for Mammalian Expression Vectors” II. Gene amplification Schematic representation of the process of selecting for an increased copy number of a gene in cultured cells. As the concentration of an inhibitor of a vital enzyme ([x]) is increased, cells with extra copies of the gene that encodes this enzyme survive. The increments of the gene copy number occur by chance among thousands of cells. The circles indicate cells; the numerals indicate numbers of gene copies. 8  How to select and amplify the genes of transfected cells “Selectable Markers for Mammalian Expression Vectors” II. Gene amplification Example: The dihydrofolate reductase–methotrexate system. Gene amplification was first demonstrated in mammalian cells by selection for resistance to methotrexate which is an analog of folic acid and competitive inhibitor of the dihydrofolate reductase enzyme, DHFR. This enzyme is essential for the conversion of the folate coenzyme required in purine and pyrimidine metabolism. Cellular resistance to the effects of methotrexate can occur by a stepwise increase in the concentration of methotrexate in the culture medium. 9  How to select and amplify the genes of transfected cells “Selectable Markers for Mammalian Expression Vectors” II. Gene amplification Cells that become resistant to methotrexate fall into one of three classes: (a) Reduced methotrexate uptake. (b) Point mutation in the dhfr gene making the enzyme less susceptible to inhibition. (c) Multiple copies of the dhfr locus allowing the production of sufficient enzyme to compete with the effect of the inhibitor. In the third class, resistance is brought about by the amplification of the gene for dihydrofolate reductase (DHFR). These resistant cells are selected by successive increases in the dosage of methotrexate. The cells can have highly amplified dhfr gene arrays, allowing the growth of cells in concentrations of methotrexate up to ×105 the dose lethal to nonresistant wild-type cells. 10  How to select and amplify the genes of transfected cells “Selectable Markers for Mammalian Expression Vectors” II. Gene amplification Example: The dihydrofolate reductase–methotrexate system. Briefly, dihydrofolate reductase catalyzes the reduction of dihydrofolate to tetrahydrofolate, which is required for the production of purines. Methotrexate is a competitive inhibitor of dihydrofolate reductase. Sensitivity to methotrexate can be overcome if the cell produces excess dihydrofolate reductase, and as the methotrexate concentration is increased over a period of 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. 11  How to select and amplify the genes of transfected cells “Selectable Markers for Mammalian Expression Vectors” Examples For the most part, the systems that are used to select transfected mammalian cells are the same as those for other eukaryotic host cells. 12  How to get genes to express proteins The insertion of DNA into a cell does not guarantee gene expression and synthesis of the corresponding protein. DNA transcription requires a number of expression signals which are specific for mammalian cells. The expression signals are usually added to the gene before it is transfected into a cell and is referred to as an ‘expression system’. The efficiencies of expression vary and depend upon the gene, expression signals and the cell line used. Stable gene expression is dependent upon the integration of a segment of DNA into the nucleus of the host cell line or an efficient extrachromosomal replication system that is maintained during cell division. Depending upon the method used, gene expression may be transient or stable. 13  How to get genes to express proteins Expression cassettes A number of expression signals are needed to ensure that a gene is expressed in mammalian cells. These signals are common to a wide variety of genes and can be used in a range of cell lines. An expression cassette is a DNA sequence containing these signal sequences that is incorporated into a vector used to transfect mammalian cells. 14  How to get genes to express proteins Expression cassettes The signal sequences of an expression cassette include the following. A promoter that is required to initiate transcription of the gene. This marks the point at which transcription of a gene starts following RNA polymerase binding. Promoters contain highly conserved sequences (for example, the ‘TATA’ box or ‘initiator’) located upstream of the initial site of transcription. Promoters taken from an animal virus (for example, SV40, adeno- or retroviruses) have been used to express a number of transfected genes. These are described as ‘strong promoters’ because they allow a high rate of transcription. A ribosome-binding site is a short nucleotide sequence required for attachment of mRNA to ribosomes. A terminator is required downstream of the expressed gene to mark the point at which transcription stops. 15  How to get genes to express proteins I. Transient gene expression This is generally used as a preliminary test of gene expression. The production of a protein from a transfected cell is assayed from a sample of the cell culture after a short period of time—usually 1–3 days following the uptake of DNA. For this, the transfected cells need not be selected or isolated but the rate of incorporation and expression of DNA in the cells needs to be high. However, the cells are not genetically stable and soon lose their expression ability. The most commonly used transient expression system involves COS cells. 16  How to get genes to express proteins II. Stable gene expression Stable expression systems are used to construct and isolate transfected cells that are genetically stable and are capable of indefinite synthesis of a particular protein. Recombinant vectors can be constructed that maximize the chances of obtaining such cells. The limiting factor in obtaining stable transfected cells is usually the frequency of DNA integration, rather than the frequency of cellular uptake. The ideal recombinant cell is one that grows with a doubling time of ~18 h, is genetically stable and secretes a recombinant protein at ~20% of total cellular protein. The most commonly used cell line for the stable expression of a recombinant protein is the Chinese hamster ovary (CHO) line— primarily because the cells have been well studied and characterized. 17  Plasmid Integration and Chromosomal Environment A major consideration for high levels and long-term stability of heterologous-protein production is the site of integration of the gene of interest into the mammalian cell chromosome. Expression of high levels of protein from plasmid vectors is transient and inevitably results in loss of the vector, which cannot be propagated in mammalian cells, or death of the host cell. 18  How to get genes to express proteins Plasmid Integration and Chromosomal Environment 19  Plasmid Integration and Chromosomal Environment Stable cell lines in which the target gene is integrated into the chromosome have been generated to overcome this problem. However, the site of integration can have a significant impact on the levels of target protein produced. While much of the genome is highly condensed (heterochromatin) and contains silent genes or genes with low levels of expression, other regions are less condensed (euchromatin) and contain actively transcribed genes. For enhanced expression and stability, the target gene should be integrated into euchromatin, rather than heterochromatin. Because a larger portion of the genome is in the heterochromatin form, there is a greater chance that the target gene will be inserted into one of these regions. 20  How to get genes to express proteins Plasmid Integration and Chromosomal Environment Therefore, genetic engineering strategies to prevent the surrounding DNA from decreasing transcription of inserted genes are being explored. These strategies exploit natural cellular processes (known as epigenetic modifications) that contribute to the dynamic state of chromatin. Chromatin structure, that is, the degree of DNA packing, is altered in two general ways. ✦Histone proteins are modified by addition of chemical groups, such as an acetyl group, to specific amino acids. ✦ By insertion of chromatin-relaxing DNA elements. Such as, inserting stabilizing and antirepressor (STAR) element on both sides of the expression cassette to block repression & Ubiquitously acting Chromatin-Opening Elements (UCOE) Chromatin-modifying elements that prevents epigenetic silencing, thereby maintaining the genes in an open state for transcription to occur. 21  How to get genes to express proteins Plasmid Integration and Chromosomal Environment Techniques to relax chromatin structure and thereby increase the expression of introduced genes include modifying host strains to express proteins that alter chromatin structure at the site of vector integration or inserting DNA elements that prevent chromosome condensation together with the target gene. Increased histone acetylation, which leads to increased gene transcription, can be accomplished either by increasing the expression of histone acetyltransferase or by decreasing the activity of histone deacetylase. Strategies to increase expression of recombinant proteins in mammalian cells by altering chromatin structure. Local “relaxation” of chromosome condensation, which leads to increased transcription of genes in the region, can be achieved by the addition of an acetyl group to DNA-packing proteins known as histones. Histone acetylation is catalyzed by the enzyme histone acetyltransferase (HAT). 22  Engineering Mammalian Cell Hosts for Enhanced Productivity Several improvements have been made to mammalian cell lines to enhance their productivity by increasing cell growth, vector stability, gene expression, and protein secretion. Conditions in large-scale bioreactors can be stressful for mammalian cells. ✦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. Often chemical inhibitors of cell death pathways are utilized, but recently, genetic means have been explored to construct cell lines in which the apoptotic pathways are inhibited. When cells perceive a variety of stresses, an initial response is the activation of the tumor suppressor protein p53, which is a transcription factor that induces the expression of genes that encode proteins in the apoptotic pathway. 23  Engineering Mammalian Cell Hosts for Enhanced Productivity Apoptotic pathway inhibition One method to improve cell growth and viability under culture conditions in bioreactors is to prevent p53 from activating the cell death response pathway. How? ‣ The mouse double-mutant 2 protein (MDM2) binds to protein p53 and prevents it from acting as a transcription factor. ‣ MDM2 also marks p53 for degradation. ‣ HEK 293 and 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. 24  Engineering Mammalian Cell Hosts for Enhanced Productivity “Apoptotic Pathway Inhibition” FIGURE : Strategy to Inhibit apoptotic pathways 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. 25 Engineering Mammalian Cell Hosts for Enhanced Productivity “Overcoming the effect of oxygen consumption” 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. To counteract the acidification of the medium from lactate secretion, alkaline compounds are typically added; however, they also contribute to reduced cell growth by increasing the osmolality of the medium. A more effective approach may be to either decrease the expression of lactate dehydrogenase or increase the expression and/or the activity of pyruvate carboxylase in host cells. The human pyruvate decarboxylase gene was cloned into an expression vector under the control of the CMV promoter and the SV40 polyadenylation signals and transfected into CHO cells. 26 Engineering Mammalian Cell Hosts for Enhanced Productivity “Overcoming the effect of oxygen consumption” When oxygen is present, pyruvate 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. 27 Engineering Mammalian Cell Hosts for Enhanced Productivity “Cell secretory system” Many heterologous 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. 28  Engineering Mammalian Cell Hosts for Enhanced Productivity “Cell secretory system” 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. - A more effective strategy may be to simultaneously over- express several, if not all, of the proteins that make up the secretory mechanism. - Simultaneous up-regulation of the genes encoding these proteins can be achieved through the enhanced production of the transcription factor X box protein 1 (Xbp-1), a key regulator of the secretory pathway. - The expression of chaperones and other proteins of the secretion apparatus is controlled by the transcription factor Xbp-1. 29  Engineering Mammalian Cell Hosts for Enhanced Productivity “Cell secretory system” FIGURE: Strategy to increase yields of secreted recombinant proteins from mammalian cells by simultaneously up regulating the expression of several proteins in the secretion apparatus. (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 cleaves the transcript to yield mature xbp-1 mRNA (the red and blue boxes represent exons) that is translated into a stable, functional transcription factor. (C) Recombinant CHO cells were transfected with a truncated gene including only the xbp-1 exons and overproduced a functional Xbp-1 transcription factor that directed the production of high levels of proteins required for protein secretion. 30  Regulation of gene expression The ability to regulate the expression of a foreign gene by the addition of an external stimulus can have distinct advantages. In some cases, over expression of a specific protein can be growth inhibitory or toxic to cells. So, it would be an advantage to switch off gene expression during cell growth and only allow the foreign gene to be expressed when maximum cell density is attained. This type of switching mechanism requires a regulated promoter such as the one isolated from mouse mammary tumor virus, which can be induced by dexamethasone. The inducible promoter can be used to induce the transcription of any gene associated with it in an expression vector when dexamethasone is added to the culture medium. The presence of the promoter can also enhance the level of protein expression. 31 Conclusion In sum, mammalian cell expression systems are as versatile and effective as other eukaryotic expression systems. However, industrial production of a recombinant protein with engineered mammalian cells is costly. Consequently, less expensive expression systems are favored unless authenticity of an important recombinant protein can be obtained only with mammalian cells. Consequently, expression systems were devised for fungal, insect, and mammalian cells. With respect to the ease and likelihood of obtaining an authentic protein from a cloned gene, each of these systems has distinct merits and shortcomings. In other words, there is no single eukaryotic host cell that is capable of producing an authentic protein from every cloned gene. All eukaryotic expression vectors have the same basic format. 32 Summary 33 Summary 34 Summary 35 Summary A number of heterologous proteins have been successfully synthesized in prokaryotic host cells. However, many proteins require eukaryote-specific post translational modifications, such as glycosylation, to be functional. The gene of interest, which may be equipped with sequences that facilitate the secretion and purification of the heterologous protein, is under the control of eukaryotic promoter and polyadenylation and transcription terminator sequences. Many therapeutic proteins that require a full complement of Post- translational modifications are now produced in cultured mammalian cells, such as CHO cells. Most of the vectors that have been developed to introduce foreign genes into mammalian cells are based on mammalian viruses, especially SV40. The viral genome has been altered to remove some viral genes required for replication and viral-protein production and to include suitable mammalian transcription and translation signals to drive expression of the cloned gene. A major challenge for production of high levels of heterologous proteins in mammalian cell lines is preventing cell death, which is often induced by the stressful conditions of large-scale bioreactors. 36 References Glick, Bernard R. Molecular biotechnology : principles and applications of recombinant DNA / Bernard R. Glick, Jack J. Pasternak, and Cheryl L. Patten. — 4th ed. (chapter 7 heterologous protein production in eukaryotic cells, “Mammalian cell expression systems” Michael Butler, Animal Cell Culture and Technology, Second Edition, (chapter 6) https://www.news-medical.net/whitepaper/20211119/The-Challenges-Faced- in-Recombinant-Protein-Expression.aspx https://www.sigmaaldrich.com/EG/en/technical-documents/technical-article/ protein-biology/protein-expression/protein-expression-systems 37