Molecular Biotechnology: Principles and Applications - Glick, B. R., & Pasternak, J. J. 2002

Fundamentals of Molecular Biotechnology
Production of Recombinant Proteins Using Eukaryotic Systems
Saccharomyces cerevisiae Expression Systems

The conventional Yeast Saccharomyces cerevisiae is widely used for the Expression of cloned eukaryotic genes for several reasons. First, it is a unicellular Organism whose genetics and physiology have been studied in detail, and it can be cultured in both small laboratory flasks and industrial bioreactors. Second, several strong promoters from this yeast have been isolated and characterized, and natural 2-µm Plasmids can serve as the basis for endogenous yeast expression vector systems. Third, S. cerevisiae Cells perform a wide range of post-translational modifications. Fourth, very few of the yeast's own Proteins are secreted into the medium; thus, if a heterologous protein is secreted by The Cell, its purification is greatly simplified. Fifth, because yeast has been used for centuries in baking and brewing, the U.S. Food and Drug Administration (FDA) has classified S. cerevisiae as "generally recognized as safe" (GRAS). Consequently, utilizing these organisms to produce medically applicable proteins circumvents the extensive safety testing required for unapproved microorganisms. Several proteins synthesized in S. cerevisiae are already used as Vaccines, Pharmaceuticals, and Diagnostics (Fig. 7.2).

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Fig. 7.2. Recombinant proteins synthesized in S. cerevisiae expression systems. HIV-I, HUMAN IMMUNODEFICIENCY VIRUS type 1.

Vectors for S. cerevisiae

There are Three types of Expression Vectors for S. cerevisiae: 1) episomal, or Plasmid Vectors; 2) integrative vectors; and 3) yeast artificial Chromosomes (YACs). Plasmid vectors have been widely used to produce both secreted and non-secreted heterologous proteins. However, plasmid-based expression systems often prove unstable when cells are grown in large volumes (>10 L). The second vector strategy has not yet achieved widespread use, despite the fact that Integration of the expression vector or transcripton into chromosomal DNA yields a stable recombinant organism. The limitation here is that the copy number of the cloned Gene is restricted to one per chromosome, resulting in a low final protein yield. Although tandem gene sequences could theoretically be used, they frequently prove unstable. Therefore, researchers have focused on single-gene plasmid vectors while modifying growth conditions to enhance plasmid stability.

Yeast artificial chromosomes (YACs) are designed to clone large DNA fragments (100 kb), which are then maintained within the yeast cell as independent chromosomes. The YAC system is exceptionally stable. It has been instrumental in the Physical Mapping of The Human Genome and the analysis of large transcriptons, as well as the Construction of Genomic libraries containing individual Human chromosomes. A YAC vector resembles a natural chromosome because it contains a sequence functioning as a METABOLISM/36.html">DNA Replication origin (an autonomously replicating sequence), a segment of the yeast centromeric region, and sequences generated at both ends during DNA linearization that act as telomeres to ensure chromosomal stability (Fig. 7.3). The insertion of foreign DNA into a YAC can disrupt the reading frame of a yeast marker gene. Consequently, the product of this gene is not formed, resulting in a colorimetric reaction when cells are grown on specialized media. Furthermore, some YAC vectors carry a selectable marker independent of the cloning site. Despite all their advantages, YACs have not yet been utilized for the industrial synthesis of heterologous proteins.

Direct expression in S. cerevisiae The term "direct expression" refers to vector systems in which the synthesized proteins accumulate within the Cytoplasm of the host cell. Although various yeast expression vectors have been developed by several research groups, they share similar core features. Here, we examine the expression of a foreign gene in S. cerevisiae using the Synthesis of the human superoxide dismutase enzyme as an example.

Fig. 7.3. The YAC cloning system. The YAC plasmid (pYAC) contains an E. coli selectable marker gene (Amp^r); a replication origin functional in E. coli (oriE); and a segment of yeast DNA including the URA3, CEN, TRP1, and ARS regions (CEN, centromeric sequence; ARS, yeast autonomously replicating sequence, equivalent to a yeast replication origin; URA3, one of the uracil Biosynthesis genes; TRP1, one of the Tryptophan biosynthesis genes). T represents the telomeric Regions of the yeast chromosome, and SmaI is the cloning site. pYAC is first digested with SmaI, BamHI, and alkaline phosphatase, and then ligated with a 100-kb DNA fragment. The final genetic construct contains the cloned DNA and is stably maintained in Ura^- Trp^- yeast cells.

The superoxide anion is a byproduct of oxygen metabolism in aerobic organisms. In humans, it plays a role in stimulating the phagocytic Immune Response and directing leukocytes to sites of infection. However, excess amounts of this compound and its derivatives can cause cellular damage. The cytoplasmic enzyme Cu/Zn-superoxide dismutase (Cu/Zn-SOD) helps minimize the potential cytotoxic effects of such species; it catalyzes the dismutation of superoxide anions and hydrogen ions into hydrogen peroxide, which in turn serves as a substrate for catalase or peroxidase. Superoxide anions are also generated during the reperfusion of Organs whose Blood supply was interrupted prior to surgery. To prevent cellular damage by superoxide anions, researchers have proposed administering Cu/Zn-SOD to organs prior to reperfusion. Cu/Zn-SOD can also be used to treat inflammatory conditions such as osteoarthritis, rheumatoid Arthritis, scleroderma, and Ankylosing spondylitis. In both clinical scenarios, using a protein identical to human Cu/Zn-SOD is preferable to avoid any undesirable immune responses that might be triggered by administering an enzyme from other species.

Initially, human Cu/Zn-SOD cDNA was cloned in an E. coli expression system. However, in that system, only the initiator N-terminal Methionine was cleaved from the Cu/Zn-SOD molecule—as occurs with all proteins synthesized in E. coli—whereas the subsequent amino acid (Alanine) was not acetylated as it is in human cells. Therefore, to obtain the authentic enzyme, the human Cu/Zn-SOD cDNA was inserted into a yeast episomal vector. Because yeast cells are incapable of efficiently splicing introns, appropriate cDNAs or chemically synthesized sequences must be used to encode specific gene products. The yeast vector containing the human Cu/Zn-SOD cDNA (Fig. 7.4) comprised: 1) the yeast leucine biosynthesis gene (LEU2); 2) a 2-µm plasmid segment containing a yeast DNA replication initiation signal, which ensures plasmid replication within yeast cells; 3) a selectable marker—the E. coli ampicillin resistance gene (Amp^r)—and an E. coli-active replication origin, enabling standard Introduction/32.html">Genetic Engineering Procedures required for plasmid construction in E. coli cells; and 4) human Cu/Zn-SOD cDNA inserted between the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase gene (GAPDp) and a sequence containing the Transcription termination and mRNA polyadenylation signals of the same gene (GAPDt).

A leucine-deficient yeast strain (LEU2^-) was transformed with this vector and plated on a medium lacking leucine. Under these conditions, only cells harboring the functional LEU2 gene located on the vector can grow. The GAPD promoter is unregulated, and transcription from it occurs continuously. Consequently, the human Cu/Zn-SOD cDNA is transcribed constitutively throughout the growth phase. In this experiment, large amounts of Cu/Zn-SOD accumulated in the yeast cells, with the amino group of the N-terminal alanine residue being acetylated, just like the native protein from human cells.

Fig. 7.4. An S. cerevisiae expression vector. The human Cu/Zn-SOD cDNA is inserted between the promoter (GAPDp) and the termination-polyadenylation signal (GAPDt) of the S. cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene. The LEU2 gene, inserted into the middle of the yeast 2-µm plasmid, encodes one of the Enzymes of leucine biosynthesis. The ampicillin resistance gene (Amp^r) and the E. coli replication origin (ori^E) were recloned from plasmid pBR322.

Secretion of heterologous proteins synthesized by S. cerevisiae

In yeast cells, only secreted proteins undergo glycosylation; therefore, production of recombinant proteins that require N- or O-glycosylation for their active conformation necessitates secretion systems. To achieve this, the so-called pre-pro-α-factor—the leader (signal) sequence of the yeast α-mating factor gene—must be placed upstream of the cDNA encoding the protein of interest. This ensures that the synthesized recombinant protein is efficiently secreted by the yeast.

During transit through the secretory pathway, the protein undergoes disulfide bond formation, proteolytic Cleavage, and other post-translational modifications, meaning that the fully active protein is sometimes released directly into the medium. The leader peptide directs translocation across the cytoplasmic membrane and drives secretion, after which it is cleaved by a yeast endoprotease that recognizes the Lys-Arg dipeptide. Consequently, the Lys and Arg codons must be positioned immediately upstream of the cDNA so that, following signal peptide cleavage, the mature synthesized protein bears the correct amino acid residue at its N-terminus.

Utilizing an episomal expression vector containing the α-factor signal sequence, researchers successfully produced a properly modified, biologically active hirudin protein synthesized and secreted by an S. cerevisiae strain. The hirudin gene was isolated from the invertebrate Hirudo medicinalis (medicinal leech). This protein is a potent anticoagulant that does not elicit adverse immunological reactions in humans. Because it can be produced in active form in large quantities, researchers were able to readily study its efficacy in dissolving blood clots in Veins and mitigating other thrombotic complications. Unfortunately, clinical trials involving 12,142 patients, 4,131 of whom had cardiovascular diseases, revealed only marginal advantages of recombinant hirudin over heparin. These benefits are insufficient to offset the high cost of recombinant hirudin, making its widespread clinical adoption unlikely.

Further studies were undertaken to enhance the secretion efficiency of recombinant proteins by S. cerevisiae strains. Specifically, investigators tested whether overexpressing protein disulfide isomerase—a natural component of the secretory machinery that ensures proper protein folding during secretion—would boost recombinant protein yield. To this end, the yeast protein disulfide isomerase gene, placed under the control of a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter and transcription termination signal, was integrated into the S. cerevisiae chromosome. The level of protein disulfide isomerase synthesis in the engineered strain was 16-fold higher than that of the wild-type strain. Subsequently, an extrachromosomal expression vector carrying the gene for human platelet-derived growth factor B was introduced into this protein disulfide isomerase overproducing strain. The amount of human platelet-derived growth factor B secreted by this strain exceeded by 10-fold the amount secreted by the strain with normal protein disulfide isomerase levels. Protein disulfide isomerase overexpression enhances the secretion of proteins that rely on Disulfide Bonds. Modulating Other components of the yeast secretory apparatus may similarly increase the yields of recombinant proteins with different folding requirements.