Molecular Biotechnology. Bernard R. Glick

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

      Many heterologous genes are provided with a DNA coding sequence for a signal peptide that facilitates the passage of the recombinant protein through cell membranes and its release to the external environment. The main reason for this modification is that it is much easier to purify a secreted protein than one from a cell lysate. The most commonly used signal sequence for S. cerevisiae is derived from the yeast mating factor α gene. Also, synthetic signal sequences have been created to increase the amount of secreted protein. Other sequences that stabilize the recombinant protein, protect it from proteolytic degradation, and provide a purification tag can be fused onto the coding sequence of the heterologous gene. These extra amino acid sequences are equipped with a cleavage site so that they can be removed from the recombinant protein after it is purified.

      Plasmid-based yeast expression systems are often unstable under large-scale (≥10 liters) growth conditions even in the presence of selection pressure. To remedy this problem, a heterologous gene is integrated into the host genome to provide a more reliable production system. Different approaches have been devised for the integration of a cloned gene together with a selectable marker gene into an S. cerevisiae chromosome. Briefly, a functional selectable marker gene and a heterologous gene equipped with yeast-specific transcription and translation control sequences are inserted between two DNA segments derived from the ends of a nonessential yeast gene. In this instance, the integrating plasmid does not usually carry an origin of replication that functions in yeast cells. The plasmid is cleaved, and the linear fragment is transformed into S. cerevisiae. A double recombination event between homologous sequences on the linearized plasmid and a chromosome in the host inserts the piece of DNA with both target