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

Fundamentals of Molecular Biotechnology
Production of Recombinant Proteins Using Eukaryotic Systems
Other Yeast Expression Systems

Using S. cerevisiae expression systems, researchers have successfully produced A wide variety of recombinant Proteins. Unfortunately, expression yields were quite low in most cases. Furthermore, A number of other issues became apparent.

✵ Scaling up these systems often leads to plasmid loss, even when inducible promoters are employed.

✵ The heterologous protein frequently turns out to be hyperglycosylated, containing over 100 mannose residues per side oligosaccharide chain, whereas native proteins contain only 8 to 13 residues per chain. The presence of these extra mannose residues can alter the product's biological activity or its immunogenicity.

✵ In many experiments, proteins meant to be secreted instead accumulated in the periplasmic space, which further complicated their purification.

All of these challenges prompted scientists to explore The production of heterologous proteins using alternative Yeast species and eukaryotic systems. Specifically, they investigated suitable vectors—expression systems containing species-specific transcriptional and translational regulatory sequences, the feasibility of transforming these species to achieve high protein yields, and the potential for large-scale cultivation of the host Organism. Potential alternatives to S. cerevisiae include Kluyveromyces lactis, a yeast used for the industrial production of lactose (ß-galactosidase); Schizosaccharomyces pombe, a fission yeast that divides by binary fission rather than budding; Yarrowia lipolytica, which utilizes alkanes as a substrate; and Pichia pastoris and Hansenula polymorpha, both of which can use methanol as their sole source of carbon and energy.

Synthesis of the Hepatitis B Virus Surface Antigen

The methylotrophic yeast P. pastoris can be easily and cost-effectively cultivated in industrial bioreactors. Utilizing it as a host organism could significantly increase the yield of active heterologous protein products. This Conclusion is illustrated by the production of the hepatitis B virus surface antigen (HBsAg) using a specially engineered system based on an integrative vector. First, the HBsAg Gene was inserted between the alcohol oxidase 1 gene promoter (AOX1p) and the METABOLISM/31.html">Transcription termination-polyadenylation signal (AOX1t) of the same gene (Fig. 7.5). The activity of the AOX1 gene in P. pastoris is regulated by methanol. In its presence, alcohol oxidase can account for up to 30% of total cellular protein, whereas in the absence of methanol, alcohol oxidase is not synthesized at all.

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Fig. 7.5. Integrative expression vector for P. pastoris. The HBsAg gene is inserted between the promoter (AOX1p) and the transcription termination-polyadenylation signal (AOX1t) of the P. pastoris alcohol oxidase 1 gene. HIS4 is the gene encoding histidinol dehydrogenase, an enzyme involved in Histidine Biosynthesis. Additionally, the vector contains a P. pastoris origin of Replication (ori_pp), an ampicillin resistance gene (Amp^r), and an origin of replication functional in E. coli (ori^E). 3'-AOX1 is a fragment of the 3'-terminal sequence of the P. pastoris alcohol oxidase 1 gene. Arrows indicate the segment that integrates into the P. pastoris genome.

The vector (Fig. 7.5), specially designed for these studies, contained the following elements: 1) the AOX1p-HBsAg-AOX1t cassette, 2) an origin of replication functional in P. pastoris; 3) a DNA fragment containing the pBR322 plasmid origin of replication and an E. coli selectable marker; 4) a 3'-AOX1 fragment facilitating the integration of cloned DNA into a specific chromosomal site; and 5) the active histidinol dehydrogenase gene (HIS4), which encodes an enzyme involved in The biosynthesis of The amino acid histidine. The presence of pBR322 sequences in this construct allows it to be maintained in E. coli, facilitating cloning and enabling the recovery of large quantities of vector DNA when necessary.

To prevent plasmid loss, Integration of the AOX1p-HBsAg-AOX1t cassette into the P. pastoris genome was designed. To this end, a P. pastoris HIS4 strain carrying a defective histidinol dehydrogenase gene was transformed with a vector fragment containing the AOX1p-HBsAg-AOX1t, HIS4, and 3'-AOX1 elements (Fig. 7.5). As a result of a double crossover between AOX1p and 3'-AOX1 of the introduced DNA on the one hand, and the complementary sequences of the chromosomal DNA on the other, the AOX1p-HBsAg-AOX1t and HIS4 sequences were integrated into The Genome, accompanied by the deletion of the chromosomal AOX1 gene (Fig. 7.6). Cells that successfully incorporated the HIS4 gene are able to grow on histidine-free medium, a trait that can be used for their Selection. A second selection criterion is the slowed growth of cells in the presence of methanol, since following the loss of the AOX1 gene via double crossover, only the single, less efficient AOX2 gene remains active.

When grown in the presence of methanol—which induces the AOX1 promoter—clones carrying the integrated AOX1p-HBsAg-AOX1t fragment synthesized large amounts of authentic HBsAg protein, which accumulated in the Cytoplasm. The protein product formed a multisubunit complex identical to that of the corresponding protein found in human cells infected with the hepatitis B virus and reacted positively with Antibodies against this virus. Cultivating this clone in a 240-liter batch fermenter yielded enough synthesized protein for approximately 10⁷ vaccinations. Furthermore, the genetic construct remained stable throughout 200 hours of cultivation in the presence of methanol.

Fig. 7.6. Integration of a portion of the expression vector into the alcohol oxidase 1 gene of P. pastoris. A double crossover between the AOX1 gene and the AOX1p and 3'-AOX1 regions (top) leads to the integration of the vector into the genomic DNA and the deletion of the major part of the alcohol oxidase 1 (AOX1) gene in the host chromosome (bottom). The HIS4 gene product enables cells to grow on medium lacking histidine. In the presence of methanol, AOX1p drives the Transcription of the HBsAg gene, while AOX1t ensures transcription termination and polyadenylation.

Synthesis of Bovine Lysozyme C2. The ability of P. pastoris to secrete a heterologous protein was investigated using a bovine lysozyme C2 cDNA system encoding the full-length protein along with its native leader peptide. Bovine lysozyme is a gastric enzyme that degrades bacterial Cell walls; it is resistant to proteases and maintains its activity within a narrow pH range, making it suitable as a feed additive for ruminants to improve Digestion.

The vector constructed for this study was identical to the AOX1p-HBsAg-AOX1t vector described above, except that the lysozyme cDNA was inserted in place of the HBsAg coding sequence. The entire plasmid was integrated into the defective HIS4 gene copy within the P. pastoris chromosome. As a result of this integration, the bovine lysozyme gene became flanked by one active (HIS4) and one defective (HIS4^-) histidinol dehydrogenase gene (Fig. 7.7). The bovine lysozyme precursor was correctly processed in P. pastoris and secreted into the medium, with the specific activity of the secreted protein matching that of the native enzyme. High-density continuous Fermentation of a 10 L culture for 200 h yielded approximately 20 g of lysozyme.

Authentic heterologous proteins have also been produced using other yeast systems. For instance, the cDNAs encoding the a- and ß-chains of human Hemoglobin A were inserted between the methanol oxidase gene promoter (MOXp) and transcription termination signal (MOXt) of Hansenula polymorpha and arranged in tandem within an expression vector. After 40 generations, an isolate with a randomly integrated copy of the original vector was analyzed and shown to contain functional hemoglobin A with the correct tetrameric Structure: two a- and two ß-chains (a₂ß₂). Additionally, large quantities of recombinant proteins encoded by various human genes have been successfully produced using an S. pombe expression vector carrying a selectable marker gene and cloned human genes, all under the control of mammalian promoters.

Fig. 7.7. Integration of the expression plasmid vector into the defective chromosomal HIS4^- gene of P. pastoris. A crossover event between the plasmid HIS4 gene and the host cell HIS4^- gene results in the integration of the entire plasmid, which becomes flanked by functional and defective HIS4 genes. p, L, and t represent the AOX1 promoter, bovine lysozyme C2 cDNA, and the transcription termination-polyadenylation signal, respectively. The black bar indicates the defective region in the HIS4 gene.

Yeast expression systems have become vital tools for producing heterologous proteins for research, industrial, and medical Applications. However, studies have shown that no single system can guarantee the production of an authentic protein for every given gene. For this and several other reasons, Gene Expression systems utilizing insect and mammalian cells have been developed.