Activity-based approaches in protein engineering MCQs With Answer

Introduction: Activity-based approaches in protein engineering combine biochemical assays, affinity-based selections, and chemical probes to directly link enzyme function with genetic information. For M.Pharm students, understanding these strategies is crucial because they enable rational optimization of therapeutic enzymes, biocatalysts, and diagnostic proteins. This blog focuses on experimental and analytical methods—such as activity-based protein profiling (ABPP), display technologies, directed evolution coupled with functional screening, and high-throughput droplet or microfluidic assays—highlighting how activity readouts guide mutation selection and library design. Emphasis is on practical applications in formulation, stability, specificity, and catalytic efficiency relevant to pharmaceutical development and formulation science.

Q1. Which technique directly couples an enzyme’s catalytic activity to its encoding gene for selection of improved variants?

• Phage display with affinity selection only
• Activity-based display or selection methods
• Mass spectrometry of purified proteins
• Western blotting for expression level

Correct Answer: Activity-based display or selection methods

Q2. What is the main advantage of activity-based protein profiling (ABPP) in protein engineering?

• It identifies gene expression levels without activity data
• It uses covalent probes to label active-site residues and measure functional state
• It only measures protein stability under heat
• It relies solely on computational predictions

Correct Answer: It uses covalent probes to label active-site residues and measure functional state

Q3. In directed evolution, what does “iterative saturation mutagenesis” primarily aim to achieve?

• Introduce random point mutations across the whole genome
• Systematically mutate targeted residues or regions through cycles to explore combinatorial space
• Only increase expression level without changing activity
• Combine unrelated proteins into chimeras

Correct Answer: Systematically mutate targeted residues or regions through cycles to explore combinatorial space

Q4. Which display technology allows the highest-throughput screening of enzyme libraries directly for catalytic turnover using FACS-based selection?

• Yeast surface display with fluorescent product detection
• Traditional ELISA for binding
• Native PAGE separation
• Isothermal titration calorimetry (ITC)

Correct Answer: Yeast surface display with fluorescent product detection

Q5. What is the key principle behind droplet microfluidic screening for enzyme activity?

• Each droplet isolates a genotype with its phenotype enabling single-variant activity measurement
• It measures ensemble averages across large populations
• It amplifies DNA without linking to activity
• It uses 2D gel electrophoresis for separation

Correct Answer: Each droplet isolates a genotype with its phenotype enabling single-variant activity measurement

Q6. Which mutagenesis technique increases overall mutation rate non-specifically and is often used to generate diversity for activity-based selection?

• Error-prone PCR
• Site-directed mutagenesis with single base change
• Codon harmonization
• Affinity maturation without mutation

Correct Answer: Error-prone PCR

Q7. Deep mutational scanning primarily provides which of the following outputs for protein engineering?

• Quantitative maps of functional effects for thousands of single mutations
• Only three-dimensional structures of proteins
• Thermodynamic stability values for wild-type alone
• Protein concentrations in formulations

Correct Answer: Quantitative maps of functional effects for thousands of single mutations

Q8. Which approach best couples a small-molecule activity-based probe to identify active enzymes in complex mixtures?

• Activity-based protein profiling (ABPP) with covalent probes
• Surface plasmon resonance (SPR) for binding kinetics
• Native mass spectrometry without labeling
• Northern blotting for mRNA detection

Correct Answer: Activity-based protein profiling (ABPP) with covalent probes

Q9. In protein engineering, what is the purpose of using substrate analogs conjugated to reporters during selection?

• To block all catalytic activity permanently
• To produce a detectable signal only when catalyzed, linking product formation to genotype
• To denature proteins for purification
• To measure protein half-life in serum

Correct Answer: To produce a detectable signal only when catalyzed, linking product formation to genotype

Q10. Which statement best describes ribosome display in the context of activity-based selection?

• It links phenotype to genotype by forming a stalled complex of mRNA–ribosome–nascent protein, enabling selection based on binding or activity when combined with appropriate assays
• It is used only for selection of small molecules
• It requires stable secretion of full-length protein out of the cell
• It measures protein glycosylation patterns exclusively

Correct Answer: It links phenotype to genotype by forming a stalled complex of mRNA–ribosome–nascent protein, enabling selection based on binding or activity when combined with appropriate assays

Q11. Which of the following is a major limitation when using activity-based probes for enzyme discovery?

• Probes always increase enzyme activity artificially
• Probe selectivity may be limited, causing off-target labeling in complex proteomes
• Probes can only detect nucleic acids
• Probes are ineffective in any aqueous buffer

Correct Answer: Probe selectivity may be limited, causing off-target labeling in complex proteomes

Q12. What advantage does mRNA display have over phage display for enzyme engineering requiring larger library sizes?

• mRNA display links phenotype to genotype in vitro and can explore larger (>10^12) library sizes without transformation limits
• mRNA display always yields better thermostability
• Phage display does not produce any functional proteins
• mRNA display cannot incorporate noncanonical amino acids

Correct Answer: mRNA display links phenotype to genotype in vitro and can explore larger (>10^12) library sizes without transformation limits

Q13. How does “activity-based selection” differ from simple binding-based selection in display technologies?

• Activity-based selection requires readout of catalytic turnover or substrate modification, whereas binding selection measures affinity without function
• There is no difference; both measure the same property
• Binding-based selection always yields higher catalytic rates
• Activity-based selection ignores genotype linkage

Correct Answer: Activity-based selection requires readout of catalytic turnover or substrate modification, whereas binding selection measures affinity without function

Q14. Which computational method is most directly integrated with activity-based experimental workflows to prioritize variants?

• Machine-learning models trained on experimental activity datasets
• Purely ab initio quantum mechanics with no experimental input
• Random sequence generation without scoring
• GC-content optimization for PCR primers

Correct Answer: Machine-learning models trained on experimental activity datasets

Q15. What role do covalent mechanism-based inhibitors play in activity-based profiling of serine hydrolases?

• They irreversibly label active serine nucleophiles, allowing identification and quantification of active enzymes
• They always increase the hydrolase’s catalytic turnover
• They are used solely to denature proteins for SDS-PAGE
• They measure transcriptional activity

Correct Answer: They irreversibly label active serine nucleophiles, allowing identification and quantification of active enzymes

Q16. Why is coupling stability screens (e.g., thermal shift) with activity-based selection advantageous in protein engineering?

• It allows simultaneous optimization of catalytic function and formulation-relevant stability traits
• Stability always decreases catalytic activity, so coupling is not desired
• Thermal shift assays only measure DNA melting
• Activity-based selection cannot be combined with other assays

Correct Answer: It allows simultaneous optimization of catalytic function and formulation-relevant stability traits

Q17. Which of the following best describes deep sequencing’s role after activity-based selection?

• Deep sequencing quantitatively tracks variant enrichment to identify beneficial mutations and infer fitness landscapes
• Deep sequencing directly measures enzymatic turnover rates without selection
• It is only useful for measuring protein tertiary structure
• Deep sequencing replaces the need for any functional assay

Correct Answer: Deep sequencing quantitatively tracks variant enrichment to identify beneficial mutations and infer fitness landscapes

Q18. What is “consensus design” and how can it be used alongside activity-based methods?

• Consensus design uses the most common residues from homolog alignments to build stable variants, which can then be tested with activity-based screens to ensure retained function
• It randomizes all residues and ignores homology
• It guarantees maximal catalytic efficiency without testing
• Consensus design only applies to nucleic acids

Correct Answer: Consensus design uses the most common residues from homolog alignments to build stable variants, which can then be tested with activity-based screens to ensure retained function

Q19. Which readout is most suitable for high-throughput quantitative activity screening in microplate or droplet formats?

• Fluorescence-based product formation or fluorogenic substrates
• Transmission electron microscopy images
• Paper chromatography of whole-cell lysates
• Manual colorimetry with subjective scoring

Correct Answer: Fluorescence-based product formation or fluorogenic substrates

Q20. In an activity-based engineering campaign, what is a common strategy to avoid selecting for high expression rather than high specific activity?

• Normalize signal to protein amount or include counterscreens that penalize merely higher expression levels
• Always select the clones with the highest total signal without normalization
• Exclude any measurement of activity and select on mRNA levels
• Only use selection methods that cannot quantify activity

Correct Answer: Normalize signal to protein amount or include counterscreens that penalize merely higher expression levels

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