Methods for protein engineering MCQs With Answer

Introduction: This collection of MCQs on Methods for Protein Engineering is designed for M.Pharm students studying Proteins and Protein Formulations. It covers both experimental and computational strategies used to modify protein sequence, structure, stability, activity, and pharmacokinetic properties. Questions focus on directed evolution, rational design, mutagenesis techniques, display technologies, high-throughput screening, computational tools, and formulation-relevant modifications such as PEGylation and Fc-fusion. The set emphasizes principles, practical approaches, and selection/screening methods commonly used in pharmaceutical protein development, helping students consolidate theoretical knowledge and apply it to drug design and formulation challenges.

Q1. Which two broad strategies are most commonly classified under methods for protein engineering?

• Rational design and directed evolution
• Protein crystallography and NMR spectroscopy
• High-performance liquid chromatography and mass spectrometry
• Cell culture and bioreactor optimization

Correct Answer: Rational design and directed evolution

Q2. Which technique is typically used in directed evolution to introduce random point mutations across a gene?

• Error-prone PCR
• Sanger sequencing
• Site-directed mutagenesis using specific primers
• Western blotting

Correct Answer: Error-prone PCR

Q3. What is the primary purpose of site-directed mutagenesis in protein engineering?

• Introduce defined mutations at specific amino-acid positions to test hypotheses
• Randomize the entire gene for library creation
• Measure protein concentration in formulations
• Display proteins on phage particles for selection

Correct Answer: Introduce defined mutations at specific amino-acid positions to test hypotheses

Q4. Phage display is most frequently employed in protein engineering to:

• Select and affinity-mature binding proteins and peptides
• Create covalent mRNA-peptide linkages
• Perform cell-free translation of libraries
• Measure thermal stability by differential scanning calorimetry

Correct Answer: Select and affinity-mature binding proteins and peptides

Q5. Which display technology links a translated peptide covalently to its encoding mRNA, enabling genotype–phenotype linkage in a cell-free system?

• mRNA display (puromycin-mediated linkage)
• Phage display
• Yeast surface display
• Ribosome profiling

Correct Answer: mRNA display (puromycin-mediated linkage)

Q6. In error-prone PCR, which adjustment is commonly made to increase the mutation rate during amplification?

• Adding Mn2+ or increasing Mg2+ concentration
• Lowering primer concentration to zero
• Using high-fidelity DNA polymerase without modifications
• Reducing dNTP concentrations to negligible levels

Correct Answer: Adding Mn2+ or increasing Mg2+ concentration

Q7. Consensus design in protein engineering is based on which principle?

• Selecting the most frequent amino acid at each position from a multiple sequence alignment of homologs
• Randomly mutating residues until activity increases
• Using only ancestral sequences inferred from fossils
• Adding polyethylene glycol to reduce immunogenicity

Correct Answer: Selecting the most frequent amino acid at each position from a multiple sequence alignment of homologs

Q8. Which computational package is widely used for de novo protein design and predicting effects of mutations?

• Rosetta
• BLAST
• ClustalW
• Prisma

Correct Answer: Rosetta

Q9. Deep mutational scanning combines systematic mutagenesis with which technology to quantify variant effects at scale?

• Next-generation sequencing (NGS)
• Two-dimensional gel electrophoresis
• Mass spectrometry-based proteomics
• Surface plasmon resonance

Correct Answer: Next-generation sequencing (NGS)

Q10. DNA shuffling is used in directed evolution primarily to:

• Recombine segments from related parent genes to create chimeric variants
• Introduce controlled single-codon substitutions at one site
• Link phenotype to genotype via bacteriophage particles
• Measure aggregation propensity of protein variants

Correct Answer: Recombine segments from related parent genes to create chimeric variants

Q11. Ancestral sequence reconstruction in protein engineering is often pursued because ancestral proteins are frequently:

• More thermostable and tolerant to sequence changes
• Impossible to express recombinantly
• Always less active than modern proteins
• Identical in sequence to consensus designs

Correct Answer: More thermostable and tolerant to sequence changes

Q12. Which strategy is typically used for site-saturation mutagenesis when constructing codon libraries at a single position?

• Use of degenerate codons such as NNK or NNS in primers
• Performing Sanger sequencing of a wild-type gene
• Applying proteolytic digestion to remove side chains
• Using size-exclusion chromatography to separate variants

Correct Answer: Use of degenerate codons such as NNK or NNS in primers

Q13. Which engineering approach is commonly used to create new covalent constraints that can improve a protein’s thermostability?

• Introduce engineered disulfide bonds by substituting paired cysteines
• Remove all charged residues to increase hydrophobic core
• Increase PEGylation to lock tertiary structure
• Use random mutagenesis only in solvent-exposed loops

Correct Answer: Introduce engineered disulfide bonds by substituting paired cysteines

Q14. To reduce immunogenicity of a therapeutic monoclonal antibody originally from a mouse source, which method is standard?

• Humanization by grafting murine CDRs onto human framework sequences
• Increasing the protein’s isoelectric point to >10
• Adding N-linked glycans at every potential site
• Using error-prone PCR to randomize the Fc region

Correct Answer: Humanization by grafting murine CDRs onto human framework sequences

Q15. Which chemistry is frequently used for site-specific PEGylation at engineered cysteine residues?

• Maleimide-thiol conjugation
• EDC/NHS coupling to carboxyl groups
• CuAAC “click” reaction on alkyne groups only
• Schiff base formation with lysine amino groups exclusively

Correct Answer: Maleimide-thiol conjugation

Q16. In high-throughput screening for enzyme variants, which readout is commonly used for rapid activity assessment?

• Fluorometric assays with fluorescent substrates
• Transmission electron microscopy
• Single-crystal X-ray diffraction
• Isoelectric focusing gels

Correct Answer: Fluorometric assays with fluorescent substrates

Q17. Which display system is particularly useful for selecting proteins that require eukaryotic folding and post-translational modifications?

• Yeast surface display
• Phage display (M13) in E. coli only
• Ribosome display (cell-free, prokaryotic translation)
• mRNA display (cell-free)

Correct Answer: Yeast surface display

Q18. Alanine scanning mutagenesis is primarily used to:

• Identify residues critical for function by replacing them with alanine
• Increase glycosylation sites to improve solubility
• Create random libraries for directed evolution
• Attach polymers such as PEG to lysines

Correct Answer: Identify residues critical for function by replacing them with alanine

Q19. How can CRISPR/Cas systems be applied to protein engineering workflows relevant to pharmaceuticals?

• Create cell lines or organisms that express variant libraries for functional selection
• Directly edit amino acids in purified proteins in vitro
• Replace display technologies like phage display with chemical conjugation
• Measure protein melting temperatures in high-throughput

Correct Answer: Create cell lines or organisms that express variant libraries for functional selection

Q20. Which modification is commonly used to extend the serum half-life of therapeutic proteins?

• Fusion to an IgG Fc domain or albumin-binding moiety (Fc-fusion)
• Complete removal of all glycosylation sites
• Introducing multiple protease-sensitive linkers
• Reducing molecular weight below 10 kDa

Correct Answer: Fusion to an IgG Fc domain or albumin-binding moiety (Fc-fusion)

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