Table 16.1 Categories of Expressed Proteins Class of Protein Examples Expression Amount Appropriate System Enzymes and For screening: 10mg Stable insect receptors For structural Baculovirus studies: 100mg Mammalian Yeast Reagent Modifying <10mg Stable insect Baculovirus Enzyme Mammalian Yeast Therapeutic Therapeutic g/L Mammalian(CHO, Monoclonal Cytokine Hormone transmembrane, nuclear hormone, integrin), and their ligands and membrane transporters(e.g, ion channels). In basic terms, suffi cient quantities of a protein target need to be supplied in order to run the hTS. The actual amounts depend on the size of a given library to be screened and the number of hits that are obtained, which will then need to be further characterized as a rule of thumb, for purified proteins such as enzymes and receptor ligands, amounts around 10mg are usually needed to support the screen. or nonpurified proteins such as receptors, one needs to think in terms of cell number and the growth properties of the cell line. For most cell lines, screens are configured by plating between 100,000 to 300,000 cells per milliliter. By way of example, a typical screen of one million compounds in multiwell formats(e.g, 96 84, or 1536 well)could use between 0.5 to 1.5 x 10 cells.The smaller the volume of the screen, the fewer cells will be required Because protein targets require a finite amount of protein one has the flexibility of choosing from virtually any expression system. Consequently the selection of the system for producing a target protein really depends on considerations other than quantity. The most important goal is to achieve a product with the highest possible biological activity. This will enable a screen to be configured with the least amount of protein and will give the best chance of establishing a screen with the highest possible to background ratio. Other considerations include the of protein being expressed (e.g, intracellular, secreted, and membrane-associated proteins ). As discussed below, stable cell systems tend to be more amenable to secreted and membrane associated proteins, while intracellular proteins are often pro- 494 Trill et altransmembrane, nuclear hormone, integrin), and their ligands and membrane transporters (e.g., ion channels). In basic terms, suffi- cient quantities of a protein target need to be supplied in order to run the HTS. The actual amounts depend on the size of a given library to be screened and the number of hits that are obtained, which will then need to be further characterized. As a rule of thumb, for purified proteins such as enzymes and receptor ligands, amounts around 10mg are usually needed to support the screen. For nonpurified proteins such as receptors, one needs to think in terms of cell number and the growth properties of the cell line. For most cell lines, screens are configured by plating between 100,000 to 300,000 cells per milliliter. By way of example, a typical screen of one million compounds in multiwell formats (e.g., 96, 384, or 1536 well) could use between 0.5 to 1.5 ¥ 109 cells. The smaller the volume of the screen, the fewer cells will be required. Because protein targets require a finite amount of protein, one has the flexibility of choosing from virtually any expression system. Consequently the selection of the system for producing a target protein really depends on considerations other than quantity. The most important goal is to achieve a product with the highest possible biological activity. This will enable a screen to be configured with the least amount of protein and will give the best chance of establishing a screen with the highest possible signal to background ratio. Other considerations include the type of protein being expressed (e.g., intracellular, secreted, and membrane-associated proteins). As discussed below, stable cell systems tend to be more amenable to secreted and membrane￾associated proteins, while intracellular proteins are often pro- 494 Trill et al. Table 16.1 Categories of Expressed Proteins Class of Protein Examples Expression Amount Appropriate System Target Enzymes and For screening: 10 mg Stable insect receptors For structural Baculovirus studies: 100mg Mammalian Yeast Reagent Modifying <10mg Stable insect enzymes Baculovirus Enzyme Mammalian Substrates Yeast Therapeutic Therapeutic g/L Mammalian (CHO, Monoclonal myelomas) antibody (mAb) Cytokine Hormone