the vectors to the eukaryotic host for protein expression. Therefore, most eukaryotic vectors are shuttle vectors with two origins of replication and two separate selectable marker genes; one set of these genes functions in the bacterium E. coli, and the other set functions in the eukaryotic host cell. If a eukaryotic expression vector is to be used as a plasmid (i.e., as extrachromosomal replicating DNA), then it must also have a eukaryotic origin of replication. Alternatively, if the vector is designed for stable integration into the host chromosomal DNA, then it must have a sequence that is complementary to a segment of host chromosomal DNA to facilitate insertion into a chromosomal site.
Figure 3.25 Generalized eukaryotic expression vector. The major features of a eukaryotic expression vector are a eukaryotic transcription unit with a promoter (p), a multiple cloning site (MCS) in which to insert a target gene, and a DNA segment with transcription termination and polyadenylation signals (t); a eukaryotic selectable marker (ESM) gene; an origin of replication that functions in the eukaryotic host cell (orieuk); an origin of replication that functions in E. coli (oriE); and an E. coli selectable marker (e.g., Ampr) gene.
The introduction of DNA into bacterial or fungal cells is called transformation. In these systems, the term describes an inherited change due to the acquisition of exogenous (foreign) DNA. However, in animal cells, transformation refers to changes in the growth properties of cells in culture after they become cancerous. To avoid confusion, the term transfection is used to denote inherited changes in animal cells that are due to the addition of exogenous DNA.
Three techniques are commonly used to transform yeasts: electroporation, lithium acetate treatment, and cell wall removal (protoplast formation). Transfection of cultured animal cells is achieved by incubating cells with DNA that has been coprecipitated with either calcium phosphate or diethylaminoethyl (DEAE)–dextran or by electroporation. Electroporation entails subjecting cells to short pulses of electric current, thus creating transient pores through which DNA enters the cell (Fig. 2.7). Viruses, lipid–DNA complexes, and protein–DNA aggregates are also used to transfer exogenous DNA into a recipient animal cell.
Yeast Expression Systems
Yeasts share many of the molecular, genetic, and biochemical features of other, “higher” eukaryotes and are therefore a good choice for heterologous protein production. They have growth advantages similar to those of prokaryotes, such as rapid growth in low-cost medium; generally do not require growth factors to be added to the growth medium; can correctly process eukaryotic proteins; and can secrete large amounts of heterologous proteins. Initially, the yeast Saccharomyces cerevisiae was used extensively as a host cell for the expression of cloned eukaryotic genes. It has a long history of use in traditional biotechnologies in the brewing and baking industries. Today, a variety of yeast and other fungal expression systems are available, and they have been optimized for recombinant protein expression. Versatile expression vectors with broad host ranges have been constructed because the optimal host for production of a particular target protein must often be determined experimentally in a number of different systems.
High levels of recombinant protein production have been achieved using S. cerevisiae. There are advantages of using this single-celled yeast. First, a great deal is known about its biochemistry, genetics, and cell biology. The genome sequence of S. cerevisiae was completed in 1996, and it is used extensively in studies as a model organism for cell function. Second, it can be grown rapidly to high cell densities on relatively simple media in both small culture vessels and large-scale bioreactors. Third, several strong promoters have been isolated from the yeast and characterized, and a naturally occurring plasmid, called the 2µm plasmid, can be used as part of an endogenous yeast expression vector system. Fourth, S. cerevisiae is capable of carrying out many posttranslational modifications. Fifth, S. cerevisiae normally secretes so few proteins that, when it is engineered for extracellular release of a recombinant protein, the product can be easily purified. Sixth, because of its many years of use in the baking and brewing industries, S. cerevisiae has been listed by the U.S. Food and Drug Administration as a “generally recognized as safe” (GRAS) organism. It does not harbor human pathogens or produce fever-stimulating pyrogens. Therefore, the use of this organism for the production of human therapeutic agents (drugs or pharmaceuticals) does not require the same extensive experimentation demanded for unapproved host cells. A number of proteins that have been produced in S. cerevisiae are currently being used commercially as vaccines, pharmaceuticals, and diagnostic agents (Table 3.10). For example, at present, more than 50% of the world supply of insulin is produced by S. cerevisiae. Engineered S. cerevisiae strains are also major producers of a hepatitis B vaccine.
Table 3.10 Recombinant proteins produced by S. cerevisiae expression systems
| Vaccines Hepatitis B virus surface antigen Malaria circumsporozoite protein HIV-1 envelope protein |
| Diagnostics Hepatitis C virus protein HIV-1 antigens Human therapeutic agents |
| Epidermal growth factor Insulin Insulin-like growth factor Platelet-derived growth factor Proinsulin Fibroblast growth factor Granulocyte-macrophage colony- stimulating factor α1-Antitrypsin Blood coagulation factor XIIIa Hirudin Human growth factor Human serum albumin |
| HIV-1, human immunodeficiency virus type 1. |
Saccharomyces cerevisiae Expression Vectors
There are three main classes of S. cerevisiae expression vectors: episomal, or plasmid, vectors, integrating vectors, and YACs. Of these, episomal plasmid vectors have been used extensively for the production of either intra- or extracellular heterologous proteins. Typically, the vectors contain features that allow them to function in both bacteria and S. cerevisiae. An E. coli origin of replication and bacterial antibiotic resistance genes are usually included on the vector, enabling all manipulations to first be performed in E. coli before the vector is transferred to S. cerevisiae for expression.
The yeast episomal plasmid vectors are based on the high-copy-number 2μm plasmid, a small, circular plasmid found in most natural strains of S. cerevisiae. The vector replicates independently of the host chromosome via a single origin of replication (autonomous replicating sequence [ARS]), and is maintained in more than 30 copies per cell. Many S. cerevisiae selection schemes rely on mutant host strains that require a particular amino acid (histidine, tryptophan, or leucine) or nucleotide (uracil) for growth. Such strains are said to be auxotrophic because minimal growth medium must be supplemented with a specific nutrient. In practice, the vector is equipped with a functional (wild-type) version of a gene that complements the mutated gene in the host strain. For example, when a plasmid vector with a wild-type LEU2 gene is transformed into a mutant leu2 host cell and plated onto medium that lacks leucine, only cells that carry the plasmid will grow.
A number of promoters derived from S. cerevisiae genes are available for engineering efficient transcription of heterologous genes in yeast vectors (Table 3.11). Generally, tightly regulatable, inducible promoters are preferred for producing large amounts of recombinant protein at a specific time during large-scale growth. In this context, the galactose-regulated promoters respond rapidly to the addition of galactose with a 1,000-fold increase in transcription. Repressible, constitutive, and hybrid promoters that combine the features of different promoters are also available. Maximal expression also depends on efficient termination of transcription. Often, for plasmid vectors, the terminator sequence is from the same gene as the promoter.
Table 3.11 Promoters for S. cerevisiae expression vectors
