Molecular Biotechnology. Bernard R. Glick

genes into a specific chromosome site (Fig. 3.26). The plasmid DNA is linearized because DNA in this form is more likely than circular DNA to recombine with chromosome DNA. The DNA that is not integrated is lost during successive cell divisions. The major drawback of this strategy is the low yield of recombinant protein from a single gene copy.

      Figure 3.26 Integration of DNA into a S. cerevisiae chromosome. A selectable marker gene (LEU2) and a gene of interest (GOI) with transcription and translation control elements (not shown) are inserted into a yeast integrating plasmid between two segments (A1 and A2) from the ends of a nonessential yeast gene A. The ampicillin resistance (Ampr) gene and the origin of replication (oriE) function in E. coli. A leucine-requiring (leu2) yeast strain is transformed with restriction endonuclease-digested vector DNA because chromosomal DNA is more likely to recombine with linearized DNA than with circular DNA. The restriction endonuclease (RE) sites flank the segments from the nonessential gene. The DNA sequences at the ends of nonessential gene A undergo recombination (×) that leads to the incorporation of both the gene of interest and the LEU2 gene into the corresponding chromosome site. Transformants grow on medium that is not supplemented with leucine. Nonrecombined DNA is degraded.

      A YAC is designed to clone a large segment of DNA (100 kilobase pairs [kb]), which is then maintained as a separate chromosome in the host yeast cell. The YAC system is highly stable and has been used for the physical mapping of human genomic DNA, the analysis of large transcription units, and the formation of genomic libraries containing DNA from individual human chromosomes. A YAC vector mimics a chromosome because it has a sequence that acts as an origin of DNA replication (ARS), a yeast centromere sequence to ensure that during cell division each daughter cell receives a copy of the YAC, and telomere sequences that are present at both ends after linearization of the YAC DNA for stability (Fig. 3.27). In some cases, the input DNA is cloned into a site that disrupts a yeast marker gene. In the absence of the product of the marker gene, a colorimetric response is observed when recipient cells are grown on a specialized medium. Alternatively, some YAC vectors contain a selectable marker gene that is independent of the cloning site.

      Figure 3.27 YAC cloning system. The YAC plasmid (pYAC) has an E. coli selectable marker (Ampr) gene; an origin of replication that functions in E. coli (oriE); and yeast DNA sequences, including URA3, CEN, TRP1, and ARS. CEN provides centromere function, ARS is a yeast autonomous replicating sequence that is equivalent to a yeast origin of replication, URA3 is a functional gene of the uracil biosynthesis pathway, and TRP1 is a functional gene of the tryptophan biosynthesis pathway. The T regions are yeast chromosome telomeric sequences. The SmaI site is the cloning insertion site. pYAC is first treated with SmaI, BamHI, and alkaline phosphatase and then ligated with size-fractionated (100-kb) input DNA. The final construct carries cloned DNA and can be stably maintained in double-mutant ura3 and trp1 cells.

Secretion of Heterologous Proteins by S. cerevisiae

      All glycosylated proteins of S. cerevisiae are secreted. Consequently, the coding sequences of recombinant proteins that require either O-linked or N-linked sugars for biological activity must be equipped with a signal peptide to pass through the secretory system (Fig. 3.28). Usually, the signal sequence from the yeast mating type α-factor gene (prepro-α-factor) is inserted immediately in front (upstream) of the cDNA of the gene of interest. Under these conditions, correct disulfide bond formation, proteolytic removal of the signal sequence, and appropriate posttranslational modifications often occur, and an active recombinant protein is secreted. During this process, the signal peptide is removed by an endoprotease (signal peptidase) that recognizes the dipeptide lysine-arginine (Lys-Arg). The Lys-Arg codons must be located adjacent to the cDNA sequence so that, following removal of the leader peptide, the recombinant protein will have the correct amino acid at its N terminus. For example, a properly processed and active form of the protein hirudin was synthesized and secreted by an S. cerevisiae strain containing a plasmid vector that had the prepro-α-factor sequence added to the hirudin coding sequence. The gene for hirudin is from an invertebrate, the leech Hirudo medicinalis. This protein is a powerful blood anticoagulant that is not immunogenic in humans.

      Figure 3.28 Protein secretion pathway in eukaryotes. (A) A signal recognition particle (SRP) binds to the signal sequence of a secretory protein. (B) The SRP attaches to a SRP receptor on the endoplasmic reticulum (ER) membrane. (C) The secretory protein is translocated into the lumen of the ER, and a signal peptidase removes the signal sequence. (D) The secretory protein is folded, partially modified, and packaged in a transport vesicle intended for the Golgi network. (E) The ER-released vesicle carrying the secretory protein enters the Golgi network at the cis face and passes through the Golgi stack where it is further modified. After it is sorted, a plasma membrane-specific vesicle is formed at the trans face of the Golgi network. The secretory transport vesicle fuses with the plasma membrane and releases the secretory protein to the extracellular environment.

      Overexpression of proteins tends to result in the formation of undesirable intracellular aggregates of the proteins, rather than their secretion into the medium, which facilitates purification. Major problems that must be addressed to increase heterologous-protein secretion in yeast cells are the incorrect folding of the polypeptide, the activation of cellular mechanisms to cope with the stress of protein overproduction, and the aberrant processing and release of the protein of interest from the endoplasmic reticulum. Correct protein folding occurs in the endoplasmic reticulum in eukaryotes and is facilitated by a number of different proteins, including molecular chaperones, enzymes that promote disulfide bond formation, signal transduction proteins that monitor the demand and capacity of the protein-folding machinery, and proteases that clear away improperly folded or aggregated proteins (Fig. 3.29). The eukaryotic enzyme protein disulfide isomerase is instrumental in forming the correct disulfide bonds within a protein. Aberrant disulfide bond formation changes a protein’s configuration, which abolishes protein activity and causes instability. Poor yields of overexpressed proteins often occur because the capacity of the cell to properly fold and secrete proteins has been exceeded.

      Figure 3.29 Summary of protein folding in the endoplasmic reticulum of yeast cells. During synthesis on ribosomes associated with the endoplasmic reticulum (ER), nascent proteins are bound by the chaperones BiP and calnexin, which aid in the correct folding of the protein. Protein disulfide isomerases (PDI) catalyze the formation of disulfide bonds between cysteine amino acids that are nearby in the folded protein. Quality control systems ensure that only correctly folded proteins are released from the ER. Proteins released from the ER are transported to the Golgi apparatus for further processing. Prolonged binding of BiP to misfolded proteins leads to activation of the S. cerevisiae transcription factor Hac1, which controls the expression of several proteins that mediate the unfolded-protein response (UPR). Adapted from Gasser et al., Microb. Cell Fact. 7:11–29, 2008.

      Several strategies have been implemented to increase the host cell’s capacity to process higher than normal levels of proteins. The overproduction of molecular chaperones and protein disulfide isomerases may increase the yield of recombinant proteins, especially those with disulfide bonds. To test this hypothesis, the yeast protein disulfide isomerase gene was cloned between the constitutive glyceraldehyde phosphate dehydrogenase promoter and a transcription terminator sequence in a yeast integrating vector, and the entire construct was integrated into a chromosomal site. The modified strain showed a 16-fold increase in protein disulfide isomerase production compared with the wild-type strain. When protein