Expression systems in E. coli MCQs With Answer

Introduction: Expression systems in Escherichia coli form the backbone of recombinant protein production for pharmaceutical research and bioprocessing. This blog provides targeted multiple-choice questions with answers to strengthen M.Pharm students’ understanding of E. coli expression platforms. Topics covered include promoter choice, host strain selection, vector architecture, induction strategies, codon optimization, folding and solubility issues, secretion to the periplasm, fusion tags and cleavage, disulfide bond formation, and downstream implications such as endotoxin and purification challenges. Each question emphasizes conceptual depth and practical considerations used in designing, optimizing, and scaling recombinant protein production in E. coli for therapeutic and analytical applications.

Q1. Which promoter system is typically used in E. coli expression vectors to achieve very high-level transcription via bacteriophage RNA polymerase?

• T7 promoter
• lac promoter
• araBAD promoter
• trp promoter

Correct Answer: T7 promoter

Q2. Which E. coli strain is commonly used for high-level T7-driven expression because it carries the DE3 lysogen encoding T7 RNA polymerase?

• JM109
• DH5α
• BL21(DE3)
• HB101

Correct Answer: BL21(DE3)

Q3. Co-expression of molecular chaperones (e.g., GroEL/GroES) in E. coli primarily aims to:

• Increase plasmid copy number
• Promote correct folding and reduce inclusion body formation
• Enhance secretion across the outer membrane
• Stabilize mRNA transcripts

Correct Answer: Promote correct folding and reduce inclusion body formation

Q4. Which strategy is most appropriate to enable formation of disulfide bonds in recombinant proteins expressed in E. coli cytoplasm?

• Use a cold-shock promoter
• Use Origami or trxB/gor mutant strains that enhance cytoplasmic disulfide formation
• Include a strong ribosome binding site
• Co-transform with a plasmid encoding rare tRNAs

Correct Answer: Use Origami or trxB/gor mutant strains that enhance cytoplasmic disulfide formation

Q5. Which fusion tag is most useful when the goal is to improve solubility of a poorly soluble recombinant protein expressed in E. coli?

• 6xHis tag
• GST (Glutathione S-transferase)
• FLAG tag
• Myc tag

Correct Answer: GST (Glutathione S-transferase)

Q6. Placing a signal peptide (e.g., PelB) at the N-terminus of a recombinant protein directs the protein to which cellular compartment in E. coli?

• Cytoplasm
• Periplasm via the Sec pathway
• Outer membrane directly
• Cytoplasmic inclusion bodies

Correct Answer: Periplasm via the Sec pathway

Q7. Which feature of an expression vector primarily determines its plasmid copy number in E. coli?

• Promoter strength
• Origin of replication
• Antibiotic resistance gene
• Multiple cloning site length

Correct Answer: Origin of replication

Q8. Why is codon optimization often required when expressing eukaryotic genes in E. coli?

• To increase plasmid stability
• To align codon usage with abundant E. coli tRNAs and avoid translational stalling
• To enhance secretion through the Sec pathway
• To increase promoter strength

Correct Answer: To align codon usage with abundant E. coli tRNAs and avoid translational stalling

Q9. Which strain would you choose to express a protein containing multiple rare codons and why?

• BL21(DE3), because it has no rare tRNAs
• Rosetta(DE3), because it supplies tRNAs for rare codons
• DH5α, because it is recombination proficient
• Origami, because it improves mRNA stability

Correct Answer: Rosetta(DE3), because it supplies tRNAs for rare codons

Q10. What is the principal advantage of periplasmic expression over cytoplasmic expression in E. coli?

• Higher plasmid copy number
• Facilitates disulfide bond formation and simpler downstream purification due to fewer host proteins
• Faster translation rates
• Lower risk of proteolysis

Correct Answer: Facilitates disulfide bond formation and simpler downstream purification due to fewer host proteins

Q11. In the T7 expression system, what role does pLysS or pLysE play?

• Provide antibiotic resistance
• Supply rare tRNAs
• Encode T7 lysozyme to reduce basal T7 RNA polymerase activity and suppress leaky expression
• Enhance plasmid copy number

Correct Answer: Encode T7 lysozyme to reduce basal T7 RNA polymerase activity and suppress leaky expression

Q12. Which induction method allows auto-induction of protein expression without manual addition of IPTG?

• Heat shock induction
• Auto-induction media (e.g., lactose/glucose based) enabling gradual induction
• Use of lacIq alone
• Periplasmic targeting induction

Correct Answer: Auto-induction media (e.g., lactose/glucose based) enabling gradual induction

Q13. Inclusion bodies are often beneficial because:

• They always contain active, properly folded protein
• They protect recombinant proteins from proteolytic degradation and facilitate high yields and simpler initial purification
• They improve secretion to the culture medium
• They eliminate the need for downstream refolding

Correct Answer: They protect recombinant proteins from proteolytic degradation and facilitate high yields and simpler initial purification

Q14. Which downstream step is typically necessary when recovering functional protein from inclusion bodies?

• Direct chromatographic purification without further treatment
• Denaturation and refolding using optimized buffer systems and redox shuffling
• Immediate secretion to periplasm
• Use of amphipathic detergents to keep protein aggregated

Correct Answer: Denaturation and refolding using optimized buffer systems and redox shuffling

Q15. Which antibiotic marker is most commonly used for selection of pET-series expression plasmids in E. coli?

• Ampicillin (β-lactam)
• Kanamycin
• Tetracycline
• Chloramphenicol

Correct Answer: Ampicillin (β-lactam)

Q16. Which factor most directly reduces endotoxin contamination risk during downstream purification of E. coli–expressed proteins?

• Using a high-copy-number plasmid
• Efficient removal of outer membrane components using specific chromatographic steps and detergents coupled with validated endotoxin removal methods
• Inducing expression at high temperature
• Expressing protein with a 6xHis tag only

Correct Answer: Efficient removal of outer membrane components using specific chromatographic steps and detergents coupled with validated endotoxin removal methods

Q17. Which of the following best describes why IPTG is used as an inducer in lac/T7 systems?

• IPTG is metabolized to stimulate growth
• IPTG is a non-metabolizable analog of lactose that binds LacI, derepressing the promoter without being consumed
• IPTG donates phosphate groups to RNA polymerase
• IPTG increases plasmid copy number

Correct Answer: IPTG is a non-metabolizable analog of lactose that binds LacI, derepressing the promoter without being consumed

Q18. When designing an expression construct for a secreted eukaryotic protein, which consideration is least relevant in E. coli?

• Presence of appropriate signal peptide for Sec/Tat
• Codon optimization for bacterial expression
• Glycosylation patterns required for activity
• Disulfide bond pattern and redox environment

Correct Answer: Glycosylation patterns required for activity

Q19. Which protease cleavage site is commonly engineered between a fusion solubility tag and target protein to allow removal of the tag after purification?

• TEV protease recognition site
• Restriction enzyme site
• Polyadenylation signal
• Ribosomal binding site

Correct Answer: TEV protease recognition site

Q20. Leaky expression from strong promoters can be problematic for toxic proteins. Which approach helps minimize toxicity before induction?

• Use a high-copy-number origin
• Use a tightly repressible system such as lacIq plus pLysS/pLysE or switch to tightly controlled promoters (e.g., rhamnose or arabinose systems)
• Increase growth temperature prior to induction
• Avoid using any repressor elements

Correct Answer: Use a tightly repressible system such as lacIq plus pLysS/pLysE or switch to tightly controlled promoters (e.g., rhamnose or arabinose systems)

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