Introduction to Bioprocessing Notes PDF

11/11/2024, 18:02 11.1. Notes | Eduvos Eduvos (Pty) Ltd (formerly Pearson Institute of Higher Education) is registered with...

11/11/2024, 18:02 11.1. Notes | Eduvos Eduvos (Pty) Ltd (formerly Pearson Institute of Higher Education) is registered with the Department of Higher Education and Training as a private higher education institution under the Higher Education Act, 101, of 1997. Registration Certificate number: 2001/HE07/008. Date: Monday, 11 November 2024, 6:02 PM Introduction to Bioprocessing 11.1. Notes 1. Learning Outcomes By the end of this lesson, you should be able to: Describe what yeast expression systems are and why they are used in bioprocessing. Identify and differentiate between various yeast expression systems. Discuss the advantages and disadvantages associated with yeast expression. Describe the process of protein expression in yeast, from transformation to protein purification. Prescribed Reading Websites: New England Biolabs. 2024. Protein Expression in Yeast. [Online] Available at: https://www.neb.com/en/applications/protein-expression/protein-expression-in-yeast. Accessed: 25 July 2024. Merck. 2024. Protein Expression Systems. [Online] Available at: https://www.sigmaaldrich.com/ZA/en/technical-documents/technical-article/protein- biology/protein-expression/protein-expression-systems. Accessed: 25 July 2024. Online Journal Article: Roghayyeh Baghban, R., Farajnia, S., Rajabibazl, M., Ghasemi, Y., Mafi, A., Hoseinpoor, R., Rahbarnia, L., Aria, M. 2019. Yeast Expression Systems: Overview and Recent Advances. Molecular Biotechnology. 61: pp. 365–384. DOI: https://doi.org/10.1007/s12033-019-00164-8. Accessed 25 July 2024. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776235 1/13 11/11/2024, 18:02 11.1. Notes | Eduvos 2. Relevance Connection Watch the video below which demonstrates a yeast transformation practically in the lab. Look out for follow- up videos to this tutorial which demonstrate how the transformed yeast was identified: 3. Introduction Expression of proteins in yeast is a common alternative to bacterial and higher eukaryotic expression. Yeast cells offer many of the advantages of producing proteins in bacteria (such as growth speed, easy genetic manipulation, low-cost media) while offering some of the attributes of higher eukaryotic systems (such as post-translational modifications, secretory expression). Several yeast protein expression systems exist in organisms from the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula and Yarrowia. Saccharomyces cerevisiae – the work horse of yeast expression systems Preferred for its ease of use, reduced time input and costs. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776235 2/13 11/11/2024, 18:02 11.1. Notes | Eduvos 4. Yeast Expression Systems HOW? → gene of interest (target gene) inserted into a bacterial plasmid, which serves as vehicle for gene transfer and protein expression. Plasmid is then introduced into the yeast host, where it replicates and produces the desired protein. 5. Expression Vectors/ Plasmids and Selection Markers Very important aspect of this strategy is the vector system used (plasmid). Expression vectors typically contain a strong yeast promoter/terminator and a yeast selectable marker cassette. Many yeast expression vectors include the ability to optionally clone a gene downstream of an efficient secretion leader (usually that of mating factor) that efficiently directs a heterologous protein to become secreted from the cell. If a desirable protein can be produced on a large scale without the need for extraction from the cells, this will cut down on downstream bioprocessing costs. Commonly require selective markers for bacteria and yeast in the vector. Therefore, appropriate vector should have the following elements: A targeting sequence for homologous recombination/integration. A multiple cloning site for gene of interest insertion. A promoter element. A selection marker for transformation. A secretory signal for secretion of foreign proteins out of the cell. Most common type: Integration Vector/Plasmid (YIp) These are plasmids / DNA sequences that will integrate into the genome (chromosomes) of the yeast (which stands in contrast to bacteria). In contrast, Episomal Vectors/Plasmid (YEp) are also often used. An attractive feature of yeast vectors is that they can replicate in the host cell by autonomous replication (replicates without integration into the host chromosomes). https://mylms.vossie.net/mod/book/tool/print/index.php?id=776235 3/13 11/11/2024, 18:02 11.1. Notes | Eduvos Image obtained from: https://www.discoveryandinnovation.com/BIOL202/notes/lecture23.html. Both vector systems (integrative and episomal vectors) have been utilized for expression of recombinant proteins. Integrated vectors: Exhibit very high stability (essential for scalable production processes). But copy number is often low. Episomal vectors: Higher copy numbers of the expression cassette. Simple transformation protocols. But will be unstable in the absence of selection. When a yeast has been transformed with an episomal vector (containing a specific selection marker, like antibiotic resistance), the yeast lose the plasmid if the selection pressure is not maintained (if antibiotic is not added to the fermentation medium). This is because the yeast does not require antibiotic resistance if an antibiotic is not present, and so loses the plasmid. Shuttle vectors: Are able to propagate in two different host species (prokaryotic and eukaryotic). Contain an E. coli replication origin and ampicillin selectable marker. (the plasmids will contain elements of bacterial and yeast origin in order to be cloned into either species). Simplifies the transformation of yeasts. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776235 4/13 11/11/2024, 18:02 11.1. Notes | Eduvos Gene of interest inserted into the shuttle vector and introduced into the E. coli for cloning of gene into the expression vector. After selection of transformants, the construct is amplified and transformed into the yeast host. 6. Promoters for Protein Production in Various Yeast Strains Well-characterized inducible or constitutive promoters with powerful transcriptional activity are preferred for overexpression of heterologous proteins in yeast Inducible: means the promoter will be induced by a specific environmental condition. Constitutive: a promoter induced permanently, regardless of environmental factors. Great attempts have been made to develop constitutive promoters with a broad range of transcriptional activities. Inducible promoters provide the advantage of control of gene expression levels in response to the presence of particular inducer or repressor in the medium. The well-known and powerful promoters that have been used for high-level expression of foreign genes in S. cerevisiae include GAL1, GAL10, JUB1, SNR52, MET17, TDH3, TPI1, ENO1, and PDC1. See table below from Baghban et al. (2019) to see what these genes encode for and for additional promoters used in other yeast expression systems. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776235 5/13 11/11/2024, 18:02 11.1. Notes | Eduvos Image obtained from: Baghban et al. (2019). DOI: https://doi.org/10.1007/s12033-019-00164-8. 7. Creating a Yeast Expression System 1. Use of competent E. coli cells to take up DNA sequence of interest. 2. Integration of the DNA into bacterial genome or circularization of the DNA sequence to exist as a plasmid. 3. Selection of transformed E. coli using a selection marker (antibiotic). 4. Expansion of selected E. coli in appropriate culture media. (This helps to increase copy number of the plasmid and ultimately the protein of interest.) 5. Isolation of DNA or plasmid. 6. Transformation into yeast. 7. Screen the transformants for integration of DNA into yeast chromosome. 8. Selection and scaling-up of high expressing yeast clones in appropriate culture media. 9. Isolation and purification of intracellular/secreted proteins. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776235 6/13 11/11/2024, 18:02 11.1. Notes | Eduvos Steps in creating a yeast expression system in S. cerevisiae. Image obtained from: https://www.sigmaaldrich.com/ZA/en/technical-documents/technical-article/protein- biology/protein-expression/protein-expression-systems. Yeast expression systems can be divided into two groups: methylotroph and non-methylotroph. Classification of common methylotroph and non-methylotroph yeasts. Image obtained from: Baghban et al. 2019. DOI: https://doi.org/10.1007/s12033-019-00164-8. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776235 7/13 11/11/2024, 18:02 11.1. Notes | Eduvos The sub-sections that follow unpack each of these groups in more detail. 7.1. Non-Methylotrophic Yeasts Saccharomyces cerevisiae Image obtained from: https://en.wikipedia.org/wiki/Saccharomyces_cerevisiae. Preferred host over bacteria, other yeasts, and filamentous fungi in numerous physiological features related to industrial ethanol production. These features include: Tolerance to wide pH range. Tolerance to high concentrations of ethanol and sugar. Resistance to elevated osmotic pressure. One of the most commonly used eukaryotic organisms that has been utilized as a model organism (non- human species that scientists use in the lab to investigate and understand biological processes) to study the following: Gene expression regulation. Signal transduction. Aging. Apoptosis. Metabolism. Cell cycle control. Programmed cell death. Neurodegenerative diseases. Autophagy. Secretory pathways. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776235 8/13 11/11/2024, 18:02 11.1. Notes | Eduvos Important advantage of SC: Safety Has GRAS status (generally regarded as safe) (GRAS). Is non-pathogenic Historically been used in various nutritional industries and production of biopharmaceuticals. Useful products that have been produced commercially through S. cerevisiae expression systems: Hepatitis B surface antigen. Hirudin. Insulin. Glucagon. Urate oxidase. Macrophage colony-stimulating factor. Platelet-derived growth factor. Limitations to using S. cerevisiae as an expression system: Hyperglycosylation of proteins. Low protein yield. Plasmid instability. These limitations have resulted in the development of alternative expression systems including methylotrophic yeasts Pichia pastoris and Hansenula polymorpha and non-methylotrophic yeast Yarrowia lipolytica, Kluyveromyces lactis and Arxula adeninivorans. Yarrowia lipolytica Image obtained from: https://www.sciencedirect.com/topics/immunology-and-microbiology/yarrowia-lipolytica. Highly desirable for industrial purposes due to its capability to: Grow on n-paraffin. Produce high levels of organic acids (e.g. citric acid). https://mylms.vossie.net/mod/book/tool/print/index.php?id=776235 9/13 11/11/2024, 18:02 11.1. Notes | Eduvos Also used in industry to produce erythritol and lipids. Can use only limited range of C6 sugars such as glucose, fructose, and mannose. But it can utilize acetate, alcohols and hydrophobic substrates including oils, alkanes, and fatty acids. 7.2. Methylotrophic Yeasts Methylotrophic: Apart from the usual sugars (like glucose and fructose), the yeast can also utilise methanol as a sole carbon source during fermentation. (therefore, non-methylotrophic means those yeast cannot, and usually require other sources like glucose and fructose). Pichia pastoris (Recently reclassified as Komagataella pastoris) Image obtained from: https://biotechresources.com/service/pichia-pastoris/. Excellent expression host for production of heterologous proteins including industrial enzymes and biopharmaceuticals. Therapeutics that have been produced: Human erythropoietin Phospholipase C Phytase Human superoxide dismutase Trypsin Human serum albumin Collagen (promoted as a hair, nail and skin supplement) Human monoclonal antibody 3H6 Fab fragment Why is this yeast becoming a very popular alternative to S. cerevisiae? https://mylms.vossie.net/mod/book/tool/print/index.php?id=776235 10/13 11/11/2024, 18:02 11.1. Notes | Eduvos Compared to any other yeast species, P. pastoris more efficient in the secretory production of recombinant proteins. Industrial interest to this host is also attributed to a powerful methanol-regulated alcohol oxidase promoter (PAOX1). Highly efficient secretion mechanism. Posttranslational modification capabilities. High cell density growing on defined medium. Some limitations: High concentration of proteases. Difficulties in the systematic study of this yeast due to product-specific effects. Risks associated with storage and use of large amounts of methanol. Recent studies that have successfully produced recombinant proteins in P. pastoris and could be used in future: Nanobody (VHH) which works against Clostridium botulinum neurotoxin type E. A product yield of 16 mg/l was achieved that was higher than the levels produced by E. coli. Recombinant angiogenin (a potent inducer of new blood vessel formation). Adiponectin (a hormone affecting several metabolic processes). Xylanase (an enzyme that hydrolases the plant cell component xylan and that can be used in the paper and pulp industry). Hansenula polymorpha Image obtained from: https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/hansenula- polymorpha. Also able to use methanol as the only carbon and energy source. Has become popular option for heterologous protein production due to: Accessibility of its multicopy integration system. Powerful inducible promoters. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776235 11/13 11/11/2024, 18:02 11.1. Notes | Eduvos Can handle high levels of oxidative stress and heavy metals. Has thermotolerance (can perform alcoholic fermentation at high temperature and therefore, is one of the advantages of this yeast over the traditional yeast S. cerevisiae). Thermotolerance: At high temperatures (48–50°C), this naturally ferments xylose to ethanol. Products being produced: Recombinant hepatitis B vaccine Insulin Interferon alpha-2a Hirudin Phytase Hexose oxidase Lipase To end off this section, follow the provided link that will take you to a simulation entitled: “How Could You Make a Recombinant Protein Vaccine Against the New Coronavirus?”. This exercise will take you through all the steps of designing a yeast plasmid for recombinant expression in yeast to the final purification of the protein. https://www.labxchange.org/library/items/lb:LabXchange:f8187474:lx_simulation:1. 8. Advantages and Disadvantages of Using Yeast Expression Systems Advantages: Yeast exhibits rapid growth. Relatively simple to genetically manipulate. Moderately rapid expression. Works well for secreted and intracellular proteins. Less expensive. Most protein folding and post-translational modifications are possible. Fermentations for the production of proteins are easy to scale up. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776235 12/13 11/11/2024, 18:02 11.1. Notes | Eduvos High biomass production is possible. Safe, pathogen-free production. Disadvantages: Characteristic N-linked glycan structures of proteins are different when compared to the typical mammalian proteins. Requires use of yeast secretion signal peptides. https://mylms.vossie.net/mod/book/tool/print/index.php?id=776235 13/13