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Questions and Answers
What is a primary advantage of using yeast for protein expression over bacterial systems?
What is a primary advantage of using yeast for protein expression over bacterial systems?
Which yeast species is commonly referred to as the work horse of yeast expression systems?
Which yeast species is commonly referred to as the work horse of yeast expression systems?
What is a common component of yeast expression vectors that aids in gene transfer?
What is a common component of yeast expression vectors that aids in gene transfer?
Why is a selectable marker important in yeast expression vectors?
Why is a selectable marker important in yeast expression vectors?
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What feature in expression vectors helps to direct a protein for secretion from the cell?
What feature in expression vectors helps to direct a protein for secretion from the cell?
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Which of the following is NOT a requirement for an appropriate vector in yeast expression systems?
Which of the following is NOT a requirement for an appropriate vector in yeast expression systems?
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How does the use of yeast expression systems affect downstream bioprocessing costs?
How does the use of yeast expression systems affect downstream bioprocessing costs?
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What type of organism do the yeast expression systems typically come from?
What type of organism do the yeast expression systems typically come from?
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What is the primary advantage of using inducible promoters in yeast protein production?
What is the primary advantage of using inducible promoters in yeast protein production?
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Which promoter is NOT mentioned as well-known for expressing foreign genes in S. cerevisiae?
Which promoter is NOT mentioned as well-known for expressing foreign genes in S. cerevisiae?
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What is one of the first steps in creating a yeast expression system?
What is one of the first steps in creating a yeast expression system?
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What is the role of a selection marker in the transformation process?
What is the role of a selection marker in the transformation process?
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Which of the following options best describes constitutive promoters?
Which of the following options best describes constitutive promoters?
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What is the final step in the yeast expression process described?
What is the final step in the yeast expression process described?
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What process aids in increasing the copy number of the plasmid during yeast expression system creation?
What process aids in increasing the copy number of the plasmid during yeast expression system creation?
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Which step immediately follows the isolation of DNA or plasmid in the yeast expression system?
Which step immediately follows the isolation of DNA or plasmid in the yeast expression system?
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What is the most common type of vector used for secretion of foreign proteins in yeast?
What is the most common type of vector used for secretion of foreign proteins in yeast?
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What is a characteristic feature of episomal vectors?
What is a characteristic feature of episomal vectors?
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Which of the following statements is true about integrated vectors?
Which of the following statements is true about integrated vectors?
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What unique feature do shuttle vectors possess?
What unique feature do shuttle vectors possess?
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Why might a yeast transformant lose an episomal vector containing an antibiotic resistance marker?
Why might a yeast transformant lose an episomal vector containing an antibiotic resistance marker?
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What is a disadvantage of using episomal vectors in yeast?
What is a disadvantage of using episomal vectors in yeast?
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What common purpose do both integrative and episomal vector systems serve?
What common purpose do both integrative and episomal vector systems serve?
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Which of the following best describes the term 'copy number' in the context of vector systems?
Which of the following best describes the term 'copy number' in the context of vector systems?
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What is a significant characteristic of Saccharomyces cerevisiae that makes it preferred over other organisms in industrial ethanol production?
What is a significant characteristic of Saccharomyces cerevisiae that makes it preferred over other organisms in industrial ethanol production?
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Which of the following statements about Saccharomyces cerevisiae is true?
Which of the following statements about Saccharomyces cerevisiae is true?
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In which area has Saccharomyces cerevisiae NOT been utilized as a model organism?
In which area has Saccharomyces cerevisiae NOT been utilized as a model organism?
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Which of the following products is NOT listed as being produced through Saccharomyces cerevisiae expression systems?
Which of the following products is NOT listed as being produced through Saccharomyces cerevisiae expression systems?
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What is one limitation mentioned regarding the use of Saccharomyces cerevisiae in expression systems?
What is one limitation mentioned regarding the use of Saccharomyces cerevisiae in expression systems?
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What physiological feature of Saccharomyces cerevisiae contributes to its wide application in various industries?
What physiological feature of Saccharomyces cerevisiae contributes to its wide application in various industries?
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Which of the following is an example of a signal transduction pathway studied using Saccharomyces cerevisiae?
Which of the following is an example of a signal transduction pathway studied using Saccharomyces cerevisiae?
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What role does Saccharomyces cerevisiae play in the production of biopharmaceuticals?
What role does Saccharomyces cerevisiae play in the production of biopharmaceuticals?
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What is a limitation of using Saccharomyces cerevisiae as an expression system?
What is a limitation of using Saccharomyces cerevisiae as an expression system?
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Which of the following yeasts is known for its ability to grow on n-paraffin?
Which of the following yeasts is known for its ability to grow on n-paraffin?
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What are methylotrophic yeasts able to utilize as a carbon source during fermentation?
What are methylotrophic yeasts able to utilize as a carbon source during fermentation?
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What product is NOT typically produced using Pichia pastoris?
What product is NOT typically produced using Pichia pastoris?
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Which of the following is a characteristic of Yarrowia lipolytica?
Which of the following is a characteristic of Yarrowia lipolytica?
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Why is Pichia pastoris becoming a popular choice over Saccharomyces cerevisiae?
Why is Pichia pastoris becoming a popular choice over Saccharomyces cerevisiae?
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Which carbon sources can Yarrowia lipolytica NOT utilize?
Which carbon sources can Yarrowia lipolytica NOT utilize?
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What makes Kluyveromyces lactis an alternative expression system?
What makes Kluyveromyces lactis an alternative expression system?
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What is one of the main advantages of using P. pastoris for recombinant protein production?
What is one of the main advantages of using P. pastoris for recombinant protein production?
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Which recombinant protein was successfully produced in P. pastoris with a yield higher than that of E. coli?
Which recombinant protein was successfully produced in P. pastoris with a yield higher than that of E. coli?
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What is a drawback of utilizing P. pastoris in industrial applications?
What is a drawback of utilizing P. pastoris in industrial applications?
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What type of carbon source can Hansenula polymorpha utilize for growth?
What type of carbon source can Hansenula polymorpha utilize for growth?
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Which of the following characteristics is NOT associated with Hansenula polymorpha?
Which of the following characteristics is NOT associated with Hansenula polymorpha?
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What temperature range can Hansenula polymorpha ferment xylose to ethanol?
What temperature range can Hansenula polymorpha ferment xylose to ethanol?
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What is the role of the PAOX1 promoter in P. pastoris?
What is the role of the PAOX1 promoter in P. pastoris?
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What factor contributes to the industrial interest in using P. pastoris for producing recombinant proteins?
What factor contributes to the industrial interest in using P. pastoris for producing recombinant proteins?
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Study Notes
Learning Outcomes
- Students will be able to describe yeast expression systems and their use in bioprocessing.
- Students will be able to identify and differentiate between various yeast expression systems.
- Students will be able to discuss the advantages and disadvantages of yeast expression.
- Students will be able to describe the process of protein expression in yeast, from transformation to purification.
Prescribed Reading
- New England Biolabs (2024): Protein Expression in Yeast. Available at: [website address]. Accessed: July 25, 2024.
- Merck (2024): Protein Expression Systems. Available at: [website address]. Accessed: July 25, 2024.
- Roghayyeh Baghban, et al. (2019): Yeast Expression Systems: Overview and Recent Advances. Molecular Biotechnology. 61: 365–384. DOI: [DOI]. Accessed: July 25, 2024.
Introduction
- Yeast protein expression is a common alternative to bacterial and higher eukaryotic expression systems.
- Yeast cells offer advantages like growth speed, easy genetic manipulation, and low-cost media, similar to bacterial systems, while also offering post-translational modifications and secretory expression abilities.
- Several yeast expression systems exist in organisms like Saccharomyces, Pichia, Kluyveromyces, Hansenula and Yarrowia.
- Saccharomyces cerevisiae is the workhorse of yeast expression systems, preferred for its ease of use, reduced time and costs.
Yeast Expression Systems
- Gene of interest is inserted into a bacterial plasmid.
- Plasmid is introduced into the yeast host.
- Yeast replicates and produces the desired protein.
Expression Vectors/Plasmids and Selection Markers
- Important vector system for this strategy.
- Expression vectors contain a strong yeast promoter/terminator and a selectable marker cassette.
- Many yeast expression vectors can optionally clone a gene downstream of that efficiently directs a heterologous protein to be secreted from the cell.
- Selection markers help in bacterial and yeast vector selection.
- Targeting sequences, multiple cloning sites, promoters, and selection markers are key vector components.
- Integration vectors and episomal Vectors (Ylp or YEp) integrate into or replicate independently of the genome.
Yeast Cloning Vectors
- Integrative and episomal vectors use for expressing recombinant proteins.
- Integrated vectors have high stability but low copy number.
- Episomal vectors demonstrate higher copy numbers and simple transformation protocols but may be unstable in the absence of selection pressure.
- Shuttle vectors are able to propagate in both prokaryotic and eukaryotic hosts.
Promoters for Protein Production in Various Yeast Strains
- Well-characterized inducible or constitutive promoters with strong transcriptional activity are preferred for overexpressing heterologous proteins (inducible are induced by a specific environmental condition).
- Great constitutive promoters are available (e.g. GAL1, GAL10, JUB1, SNR52, MET17, TDH3, TPI1, and PDC1).
Creating a Yeast Expression System
- Use competent E. coli cells to take up DNA sequence of interest.
- Integrate DNA into bacterial genome, or circularize to become a plasmid.
- Select transformed E. coli using a selection marker (antibiotic).
- Expand selected E. coli in an appropriate culture.
- Isolate DNA or plasmid.
- Transform into yeast.
- Screen transformants for DNA integration.
- Select and scale high-expressing yeast clones.
- Isolate and purify intracellular/secreted proteins.
Yeast Classification
- Methylotrophic yeasts (e.g., Pichia pastoris, Hansenula polymorpha) utilize methanol as a carbon and energy source
- Non-methylotrophic yeasts (e.g., Saccharomyces cerevisiae) utilize the normal sugars such as glucose and fructose.
Advantages of Saccharomyces cerevisiae
- Preferred host for bacteria, other yeasts, and filamentous fungi.
- Tolerates wide pH ranges and high concentrations of ethanol and sugar.
- Resistance to elevated osmotic pressure.
- Commonly studied for gene expression regulation, signal transduction, aging, apoptosis, metabolism, cell cycle control, programmed cell death, neurodegenerative diseases, and secretory pathways.
Important Advantages and Disadvantages of Yeast Expression Systems
- Advantages
- Yeast exhibits rapid growth.
- Relatively easy to manipulate genetically
- Moderate rapid expression
- Works well for secreted and intracellular proteins.
- Less expensive
- Easy to scale up fermentations
- Disadvantages
- N-linked glycan structures differs from typical mammalian proteins
- Use of yeast secretion signal peptide is necessary.
- High biomass production is possible, and production is safe.
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Description
This quiz covers yeast expression systems and their applications in bioprocessing. Students will learn to identify and discuss various yeast systems, along with their advantages and disadvantages. Understanding the protein expression process in yeast from transformation to purification is also emphasized.
