Recombinant Protein Expression Quiz
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Questions and Answers

What is the main advantage of using the pBAD promoter in the expression of recombinant proteins?

  • It is dependent solely on glucose availability for regulation.
  • It enhances the toxicity of the production system.
  • It provides precise control of expression based on arabinose concentration. (correct)
  • It allows for continuous expression regardless of arabinose presence.

How does the presence of glucose affect the expression of the pBAD promoter?

  • It completely inhibits the promoter activity.
  • It could lower the expression due to CAP protein involvement. (correct)
  • It has no impact on the expression levels.
  • It enhances the expression of the recombinant protein.

Which factor is responsible for controlling the plasmid copy number?

  • Catabolite activator protein.
  • Transcription inducers.
  • Ribosome binding sites.
  • The rop gene. (correct)

What role do shiny Dalgarno sequences play in translation efficiency?

<p>They help the ribosome to bind the RNA and improve translation. (B)</p> Signup and view all the answers

What is the consequence of using classic codons in translation?

<p>It may result in less translation efficiency. (A)</p> Signup and view all the answers

Which factor is NOT involved in ensuring the correct folding of proteins?

<p>Environmental pH (D)</p> Signup and view all the answers

What is a potential disadvantage of secreting recombinant proteins into the periplasm?

<p>Low secretion yield (B)</p> Signup and view all the answers

What is a factor that can enhance protein folding in E. coli?

<p>Surexpression of GroE/L (D)</p> Signup and view all the answers

Which of the following contributes to the formation of insoluble inclusion bodies?

<p>Fast protein expression (C)</p> Signup and view all the answers

What method is commonly used for solubilizing inclusion bodies?

<p>Denaturing buffer with 8M urea (A)</p> Signup and view all the answers

Which modifiable factor can influence the quantity of protein produced in E. coli?

<p>Concentration of inductor (A)</p> Signup and view all the answers

What benefit do inclusion bodies provide to the cell hosting recombinant proteins?

<p>Protection against proteolysis (B)</p> Signup and view all the answers

What must a recombinant protein possess to be exported into the periplasm?

<p>Secretion signals in N-terminal (A)</p> Signup and view all the answers

What is the primary function of the pFGPD-GFP vector?

<p>To track protein localization (C)</p> Signup and view all the answers

Which promoter is associated with the pFB6 vector, used for industrial enzyme production?

<p>amyB (C)</p> Signup and view all the answers

What is a common application of the pTTTi vector?

<p>Cellulase studies (A)</p> Signup and view all the answers

Which vector is associated with heat shock promoter-driven expression?

<p>pBC-hph (C)</p> Signup and view all the answers

Which protein was mentioned as being produced in Aspergillus nidulans?

<p>Chymosin (B)</p> Signup and view all the answers

What type of resistance does the hygR gene provide in pAN7-1?

<p>Antibiotic resistance (C)</p> Signup and view all the answers

Which company is implied in the production of Chymosin in Aspergillus nidulans?

<p>K. (B)</p> Signup and view all the answers

The pCAMBIA vector is primarily used for which type of studies?

<p>Functional studies (D)</p> Signup and view all the answers

What is one of the main advantages of expression systems in yeasts?

<p>Ability to perform post-transcriptional modifications (D)</p> Signup and view all the answers

Which common yeast species is primarily used for research and industrial applications?

<p>Saccharomyces cerevisiae (C)</p> Signup and view all the answers

What limitation does Saccharomyces cerevisiae present for therapeutic applications?

<p>It performs hypermannosylation, differing from human glycosylation. (B)</p> Signup and view all the answers

Why is Pichia pastoris favored for protein expression over Saccharomyces cerevisiae?

<p>It can grow to very high cell densities and yields proteins effectively. (B)</p> Signup and view all the answers

What post-translational modifications can Saccharomyces cerevisiae perform?

<p>Glycosylation, phosphorylation, and disulfide bond formation (A)</p> Signup and view all the answers

What carbon source does Pichia pastoris utilize for growth?

<p>Methanol (C)</p> Signup and view all the answers

What does GRAS stand for in relation to Saccharomyces cerevisiae?

<p>Generally Recognized As Safe (B)</p> Signup and view all the answers

What makes expression systems in filaments fungi and yeasts particularly advantageous?

<p>Perform various necessary post-translational modifications (A)</p> Signup and view all the answers

What is the main characteristic of constitutive expression in integrative vectors?

<p>The gene integrates into the host genome for stable expression. (B)</p> Signup and view all the answers

Which promoter is commonly used to drive constitutive expression in mammalian cells?

<p>CMV promoter (D)</p> Signup and view all the answers

What is the difference between integrative vectors and replicative vectors?

<p>Integrative vectors provide stable expression while replicative vectors offer transient expression. (A)</p> Signup and view all the answers

Why are selection processes necessary when using integrative expression vectors?

<p>To select only the cells that have integrated the vector. (A)</p> Signup and view all the answers

What is a significant drawback of using integrative and replicative vectors?

<p>They require more time and effort to establish. (A)</p> Signup and view all the answers

What is the purpose of using Neonucin in the selection process for integrative expression?

<p>It provides a positive selection for cells that have integrated the vector. (D)</p> Signup and view all the answers

Which statement is true about the use of replicative vectors?

<p>They are best for high-yield, short-term expression. (B)</p> Signup and view all the answers

What happens to cells when transfected with a constitutive expression vector?

<p>Only cells that successfully integrate the vector will survive under selective conditions. (B)</p> Signup and view all the answers

What is the main purpose of the T7 promoter in the pET expression system?

<p>To initiate transcription of recombinant proteins. (A)</p> Signup and view all the answers

What limitation does the E.coli BL21 strain face when expressing eukaryotic proteins?

<p>It does not recognize certain rare codons. (D)</p> Signup and view all the answers

What is the role of rare tRNA plasmids in improved BL21(DE3) strains?

<p>To facilitate the translation of rare codons found in eukaryotic genes. (D)</p> Signup and view all the answers

Why is it important for improved BL21(DE3) strains to express T7 lysozyme?

<p>To impose tight regulation on T7 polymerase expression. (D)</p> Signup and view all the answers

Which of the following codons is considered rare in E.coli and can impact protein expression?

<p>AGG (D)</p> Signup and view all the answers

What characteristic does E.coli BL21 have that affects the half-life of mRNA?

<p>It has defective RNAse E. (D)</p> Signup and view all the answers

What is the significance of having a multi-cloning site in a pET vector?

<p>To allow the introduction of multiple genes. (A)</p> Signup and view all the answers

Which component is crucial for IPTG-induced expression in bacterial host cells?

<p>T7 promoter. (A)</p> Signup and view all the answers

Flashcards

AraC protein

A regulatory protein that acts as both an activator and repressor of the arabinose operon.

pBAD promoter

A promoter that is activated by arabinose and repressed by glucose.

Expression system

A system that uses a specific promoter to control the expression of a gene of interest in bacteria.

Plasmid copy number

A regulatory element that controls the number of copies of a plasmid in a bacterial cell.

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Translation optimization

A process of optimizing the expression of a gene in a bacterial cell by modifying the sequence of the gene or the surrounding regulatory elements.

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T7 promoter

A strong promoter sequence recognized by the T7 RNA polymerase, essential for initiating transcription of the gene of interest.

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pET System

The pET (phage expression vector) system uses a T7 promoter to control the expression of a gene of interest in E.coli.

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pET vector

A vector containing the T7 promoter, lac operator, and a multi-cloning site, designed for cloning and expressing genes in E. coli.

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E. coli BL21(DE3)

A bacterial strain commonly used for protein expression, containing a copy of the T7 RNA polymerase gene under the control of the lacUV5 promoter.

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lacUV5 promoter

A specific type of promoter that is activated by IPTG, a chemical inducer. The T7 RNA polymerase gene in BL21(DE3) is under the control of the lacUV5 promoter.

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IPTG (Isopropyl β-D-1-thiogalactopyranoside)

An inducer molecule that binds to the lac repressor protein, allowing the T7 RNA polymerase gene to be transcribed, thus initiating protein expression from the gene of interest.

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T7 lysozyme

A protein inhibitor of T7 RNA polymerase, used to prevent leaky expression of recombinant proteins before induction.

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RNAse E

An enzyme that degrades mRNA. Mutations in this enzyme can improve the stability of mRNA, leading to higher protein expression.

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Protein Folding and Solubility

The process of protein folding is crucial for solubility. Proteins need to fold correctly to maintain their functional shape and interactions.

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Role of Chaperones in Folding

Molecular chaperones assist in the proper folding of proteins. Different types of chaperones, like Trigger factor, DnaK, and GroE/L, play a role in this process.

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Enhancing Folding with GroE/L

Sometimes, increasing the amount of a specific chaperone like GroE/L can enhance protein folding and solubility.

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Protein Quantity and Solubility

The amount of protein produced impacts its solubility. High protein expression can overwhelm the chaperone system, leading to misfolding and insolubility.

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Inducers and Protein Expression

Inducers like IPTG and arabinose control the level of protein expression. Adjusting their concentration influences protein production and solubility.

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Temperature and Solubility

Temperature plays a role in solubility. Changing incubation temperature can affect protein folding and solubility.

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Plasmid Copy Number and Protein Production

The number of copies of a plasmid carrying the gene for the desired protein affects its production level. Higher copy number means more protein and potential for solubility issues.

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Periplasmic Expression

The periplasm, the space between the inner and outer membrane of bacteria, is a favored location for protein expression due to its potential for reducing purification costs.

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Why are yeast expression systems advantageous?

Expression systems in yeasts are efficient platforms for producing recombinant proteins, enzymes, and biotechnological products, especially because they can perform essential post-translational modifications similar to those found in eukaryotic cells.

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What are post-translational modifications?

Post-translational modifications like glycosylation, phosphorylation, and disulfide bond formation are critical for many proteins to function correctly, particularly in eukaryotic organisms.

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What is Saccharomyces cerevisiae and why is it important in research?

Saccharomyces cerevisiae (commonly known as Baker's yeast) is a widely used organism in research and industrial applications due to its well-understood genetics and ability to perform various post-translational modifications.

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What is a limitation of Saccharomyces cerevisiae for producing therapeutic proteins?

Although Saccharomyces cerevisiae can perform glycosylation, the pattern of glycosylation it produces is often different from that found in humans. This can have implications for therapeutic applications.

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What is Pichia pastoris and what makes it a good choice for protein production?

Pichia pastoris is a methylotrophic yeast, meaning it can use methanol as a carbon source. It's particularly suited for producing high yields of protein.

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What advantage does Pichia pastoris have over Saccharomyces cerevisiae in terms of glycosylation?

Pichia pastoris has simpler glycosylation patterns compared to Saccharomyces cerevisiae, which is advantageous for producing proteins with more human-like glycosylation.

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When are yeast expression systems particularly useful?

Yeast expression systems are preferred for producing complex eukaryotic proteins that require post-translational modifications like glycosylation, which are essential for their proper function.

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Integrative Vector

A type of expression vector that integrates into the host cell's genome, resulting in stable, long-term gene expression.

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Replicative Vector

A type of expression vector that remains as an extrachromosomal entity in the host cell, offering high-yield, short-term gene expression.

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Constitutive Expression

A strong promoter that drives continuous gene expression.

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Constitutive Expression

A method of controlling gene expression where a gene is expressed continuously.

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Constitutive Expression Vector

A type of expression vector that allows for both transient and stable expression.

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Antibiotic Selection

The process of selecting cells that have successfully integrated a vector into their genome using an antibiotic.

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Transient Expression

Expression of a gene occurs for a limited time, often resulting in high levels of protein production.

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Episomal Vector

A vector that replicates independently of the host cell's genome, often used for transient expression.

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Aspergillus nidulans

A filamentous fungus commonly used for expressing recombinant proteins due to its efficient secretion system and ability to grow on cheap substrates.

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gpdA promoter

A strong promoter in Aspergillus nidulans often used to drive high levels of gene expression.

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hygR

A gene conferring resistance to hygromycin B, commonly used as a selectable marker in Aspergillus nidulans.

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cbh1 gene

A cellobiohydrolase gene, often used to express the enzyme cellobiohydrolase in different fungi.

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pAN7-1 vector

A common vector system used in Aspergillus nidulans for expressing recombinant proteins, often containing the gpdA promoter and hygromycin resistance gene.

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cbh2 promoter

A well-established promoter commonly used in Trichoderma reesei, often used in the production of cellulases.

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pCAMBIA vector

A vector often used for expressing genes in fungal systems, commonly used for functional studies in plants and fungi.

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Chymosin

A protein (chymosin) commonly used for cheese production, often expressed in Aspergillus nidulans.

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Study Notes

Recombinant Protein Production and Purification

  • The general scheme for producing and purifying recombinant proteins involves four key steps:

    • Constructing an expression vector.
    • Selecting and culturing a host cell.
    • Production of the protein.
    • Extracting, separating, and purifying the protein.

  • The order of these steps generally follows a reverse order strategy. First designing the purification method, then the production strategy, selection of the host cell, and finally choosing the expression vector.

Host-Vector Systems

  • Important considerations for choosing a host and vector include:
    • The intended use of the protein (e.g., pharmaceutical, industrial). This influences the required features, such as purity, quantity and safety standards.
    • The characteristics of the protein. Aspects like stability, solubility and post-translational modifications (e.g., glycosylations) determine a suitable system.
    • Quantitative and economic criteria, such as the expression level (yield) and optimization of purification processes. Equipment should also be considered for cost-effectiveness.

Construction of an Expression Vector

  • Recombinant protein production begins by constructing an expression vector. This involves taking the genetic information (gene) of interest from a donor organism, and inserting it into an expression vector.
  • This process may involve multiple steps like: restriction of nucleases to isolate the gene of interest, amplification of the gene via PCR, insertion of the gene of interest into the vector, and finally sealing the vector.
  • Critical components of this process include: origin of replication, selectable marker, reporter gene and fusion tags (optional).
  • The transgene, the gene of interest, is a chimeric (non-natural) construct, different from natural genes.
  • Typical transgene components include promoter regions (to initiate transcription), open reading frame (ORF - coding sequence), and terminator sequences (for transcription termination).
  • The choice of promoter sequences determines if expression is constitutive or inducible.

Expression Systems in Bacteria

  • Escherichia coli (E. coli): is the most ancient system, and is often the least expensive choice for research and production.
  • Key features of E. coli include: The least expensive approach, used widely for research and production, but is limited to simple proteins, and with little or no post-translational modifications.
  • Plasmids (extrachromosomal circular DNA, 2-6 kbp), are commonly used vectors. These have variable copy numbers (controlled by rop or par genes) and often include antibiotic resistance genes for selection.
  • The process involves introducing a plasmid with the gene of interest into the bacteria, followed by selection using antibiotic resistance markers.
  • Important characteristics of bacterial strains include recA- (reduced recombination), and dam-/dcm- (mutated cytosine methyltransferases), to minimize the risk of plasmid and/or bacterial genome manipulation. This reduces the risk of protein inactivation and host cell damage.

Expression Systems in Yeasts and Filamentous Fungi

  • Yeasts (e.g., Saccharomyces cerevisiae, Pichia pastoris): and filamentous fungi (Aspergillus niger) are used for protein production due to their ability to carry out post-translational modifications (e.g., glycosylation, phosphorylation, folding), which are usually needed for functional eukaryotic proteins.
  • Saccharomyces cerevisiae is a commonly used yeast due to its well-understood genetics, suitability for pharmaceutical production and good protein yields. It is also generally recognized as safe (GRAS)
  • Pichia pastoris, a methylotrophic yeast that uses methanol as a carbon source. Protein production is often driven by the AOX1 promoter, an inducible promoter, which is induced by methanol.
  • Using homologous markers that are native from the cell type, offers superior expression and clear phenotype and reduces the possibility of mutation in the host cell.
  • Use of heterologous markers, are beneficial for expressing in any susceptible genetic background, however gene expression levels may vary in this type of system.

Expression Systems in Mammalian Cells

  • Mammalian cells, such as CHO cells, and HEK293 cells and their expression systems are used for protein expression, specifically for the production of eukaryotic proteins or where specific post-translational modifications are essential
  • Mammalian cells are used to produce proteins that are difficult or impossible to make in prokaryotic or yeast systems.
  • Key aspects of mammalian cell systems include complexity of protein folding, and the need for specific post-translational modifications.
  • Transient expression involves temporary introduction of the gene of interest, is useful for short-term studies, has a lower cost and faster setup.
  • Stable expression involves integration of the gene into the host genome, is useful for large-scale production or therapeutic applications, and requires specialized screening and selection processes.

Further Considerations for Vector and Host Selection

  • Expression vectors, and host selection for specific needs influence protein production. Different expression vectors can be integrated for specific purposes, like plasmids.
  • Regulation of expression (e.g., using promoters or inducible promoters), is a critical element in controlling protein production.
  • Codon usage in the gene of interest, and host cell recognition of those codons is a significant consideration that affects expression and function when expressing heterologous proteins in a host.
  • The development of improved cell types and technologies ensures improved expression levels by addressing factors such as inclusion bodies or mRNA degradation.

Recombinant Protein Purification

  • Purification methods must be carefully chosen, and can be carried out by multiple steps, through dialysis for decreasing denaturing agent concentrations, or affinity chromatography to obtain the protein from denaturing agents, and eventually through column washing to release and collect the purified protein. There are more in-depth methods available to ensure optimal protein folding.
  • Correct folding of recombinant proteins is an important aspect of ensuring proper function.

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Production & Purification of Recombinant Proteins PDF

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Test your knowledge on the pBAD promoter and its applications in the expression of recombinant proteins. Explore topics such as the effects of glucose, plasmid copy number control, and translation efficiency. This quiz is essential for understanding molecular biology concepts related to protein synthesis.

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