appropriate vector and host system that is compatible with a par ticular expression need. This will largely depend on the type of protein being expressed (e.g, secreted, membrane-bound, or intracellular) and its intended use. No one system can or should be expected to meet all expression needs. n this section we will attempt to outline the critical ster involved in the planning and implementation of a successful eukaryotic expression project Planning the project will begin by answering pertinent questions such as what is known about the protein being expressed, what is its function, what is the intended use of the product, will the protein be tagged, how much protein is needed. and how soon will it be needed. Based on these con siderations, an appropriate host or vector system can be chosen that will best meet the anticipated needs. Considerations during the implementation phase of the pro- ject will include choosing the best method of gene transfer and stable selection compared to transient expression and selection methods for stable lines, and clonal compared to polyclonal selection. Finally, we will discuss anticipated outcomes from various methods, commonly encountered problems, and possible solutions to these problems. PLANNING THE EUKARYOTIC EXPRESSION PROJECT What Is the Intended use of the protein and what Quantity Is Required? Protein quantity is an important consideration, since substan tial time and effort are required to achieve gram quantities while production of 10 to 100 milligrams is often easily obtained from a few liters of cell culture. Therefore we tend to group the expressed proteins into the following three categories: target, reagent, and therapeutic protein. This is helpful both in choosing an appropri ate expression system and in determining how much is enough to meet immediate needs(Table 16.1) Targets Protein targets represent the majority of expressed proteins used in classical pharmaceutical drug discovery, which involves the configuration of a high-throughput screen(HTS) of a chemical or natural product library in order to find selective antagonists or agonists of the protein's biological activity. Protein targets include enzymes(e.g, kinases or proteases), receptors(e.g, 7 Eukaryotic Expressionappropriate vector and host system that is compatible with a par￾ticular expression need. This will largely depend on the type of protein being expressed (e.g., secreted, membrane-bound, or intracellular) and its intended use. No one system can or should be expected to meet all expression needs. In this section we will attempt to outline the critical steps involved in the planning and implementation of a successful eukaryotic expression project. Planning the project will begin by answering pertinent questions such as what is known about the protein being expressed, what is its function, what is the intended use of the product, will the protein be tagged, how much protein is needed, and how soon will it be needed. Based on these con￾siderations, an appropriate host or vector system can be chosen that will best meet the anticipated needs. Considerations during the implementation phase of the pro￾ject will include choosing the best method of gene transfer and stable selection compared to transient expression and selection methods for stable lines, and clonal compared to polyclonal selection. Finally, we will discuss anticipated outcomes from various methods, commonly encountered problems, and possible solutions to these problems. PLANNING THE EUKARYOTIC EXPRESSION PROJECT What Is the Intended Use of the Protein and What Quantity Is Required? Protein quantity is an important consideration, since substan￾tial time and effort are required to achieve gram quantities while production of 10 to 100 milligrams is often easily obtained from a few liters of cell culture.Therefore we tend to group the expressed proteins into the following three categories: target, reagent, and therapeutic protein. This is helpful both in choosing an appropri￾ate expression system and in determining how much is enough to meet immediate needs (Table 16.1). Targets Protein targets represent the majority of expressed proteins used in classical pharmaceutical drug discovery, which involves the configuration of a high-throughput screen (HTS) of a chemical or natural product library in order to find selective antagonists or agonists of the protein’s biological activity. Protein targets include enzymes (e.g., kinases or proteases), receptors (e.g., 7 Eukaryotic Expression 493