Isolation and purification of proteins MCQs With Answer

Isolation and purification of proteins MCQs With Answer

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Introduction: Isolation and purification of proteins is a critical module for M.Pharm students preparing for research or pharmaceutical development. This collection of MCQs focuses on theoretical principles and practical techniques used to extract, concentrate, and purify proteins from biological sources while preserving activity and stability. Questions cover cell disruption, fractionation, chromatographic methods (ion-exchange, affinity, size-exclusion, hydrophobic interaction), electrophoretic separation, ultrafiltration, dialysis, protein refolding, analytical characterization, and process considerations such as yield, purity, and scale-up. These targeted MCQs, with answers, will help reinforce core concepts and prepare students for examinations and laboratory applications.

Q1. Which protein isolation method primarily exploits differences in protein solubility as a function of salt concentration?

  • Ion exchange chromatography
  • Size exclusion chromatography
  • Salting out (ammonium sulfate precipitation)
  • Affinity chromatography

Correct Answer: Salting out (ammonium sulfate precipitation)

Q2. Which cell disruption technique uses sound waves to fragment cell walls and membranes, commonly used for bacterial and yeast cells?

  • French press
  • Sonication (ultrasonic disruption)
  • Bead milling
  • Detergent lysis

Correct Answer: Sonication (ultrasonic disruption)

Q3. In differential centrifugation, which factor is NOT directly changed to separate cellular components?

  • Relative centrifugal force (RCF)
  • Rotor type (fixed-angle vs swing-out)
  • Buffer ionic strength
  • Spin time

Correct Answer: Buffer ionic strength

Q4. Which chromatographic technique separates proteins based primarily on their net surface charge at a given pH?

  • Size-exclusion chromatography
  • Ion-exchange chromatography
  • Hydrophobic interaction chromatography
  • Affinity chromatography

Correct Answer: Ion-exchange chromatography

Q5. Which elution strategy is typically used to release a His-tagged recombinant protein from a Ni-NTA affinity resin?

  • High pH buffer
  • High salt (2 M NaCl)
  • Imidazole gradient
  • Organic solvent (acetonitrile)

Correct Answer: Imidazole gradient

Q6. Which statement best describes size-exclusion chromatography (gel filtration)?

  • Proteins bind to charged groups and are eluted by salt gradients
  • Large proteins enter pores and are retained longer than small proteins
  • Separation is based on hydrophobic patches interacting with matrix
  • Smaller molecules penetrate porous beads and elute later than larger molecules

Correct Answer: Smaller molecules penetrate porous beads and elute later than larger molecules

Q7. Hydrophobic interaction chromatography (HIC) typically requires which preparatory condition for proteins before loading onto the column?

  • Low ionic strength buffer to increase hydrophobic interactions
  • High concentration of chaotropic agents (e.g., urea)
  • High salt concentration to enhance hydrophobic interactions
  • Reducing agents to maintain disulfide bonds

Correct Answer: High salt concentration to enhance hydrophobic interactions

Q8. Which analytical method provides information about the molecular weight distribution and purity under denaturing conditions?

  • Native PAGE
  • SDS-PAGE
  • Isoelectric focusing (IEF)
  • Size-exclusion HPLC under native conditions

Correct Answer: SDS-PAGE

Q9. Isoelectric focusing separates proteins based on which property?

  • Hydrodynamic radius
  • Net charge at a specific pH (isoelectric point)
  • Affinity for specific ligands
  • Hydrophobic surface area

Correct Answer: Net charge at a specific pH (isoelectric point)

Q10. When designing a purification protocol, which metric expresses the ratio of specific activity after and before purification?

  • Total yield (%)
  • Specific activity
  • Purification factor (fold purification)
  • Recovery index

Correct Answer: Purification factor (fold purification)

Q11. Which detergent is commonly used to solubilize membrane proteins while preserving tertiary structure for purification?

  • SDS (sodium dodecyl sulfate)
  • Triton X-100 or DDM (n-dodecyl-β-D-maltoside)
  • Guanidine hydrochloride
  • Urea

Correct Answer: Triton X-100 or DDM (n-dodecyl-β-D-maltoside)

Q12. Which method is most appropriate for concentrating a protein solution without causing significant denaturation?

  • Lyophilization directly from low-concentration buffer
  • Repeated freeze-thaw cycles
  • Ultrafiltration using appropriate molecular weight cutoff membrane
  • Boiling to reduce volume

Correct Answer: Ultrafiltration using appropriate molecular weight cutoff membrane

Q13. Dialysis is primarily used to accomplish which of the following in a protein purification workflow?

  • Increase protein molecular weight
  • Exchange buffer and remove small-molecule impurities
  • Separate proteins by charge
  • Sterilize the protein solution

Correct Answer: Exchange buffer and remove small-molecule impurities

Q14. Which approach is most effective to remove endotoxin (lipopolysaccharide) from purified recombinant proteins?

  • High-salt dialysis
  • Polymyxin B affinity chromatography or endotoxin-specific removal resins
  • Heating to 95°C for 10 minutes
  • Size-exclusion chromatography alone

Correct Answer: Polymyxin B affinity chromatography or endotoxin-specific removal resins

Q15. During protein refolding from inclusion bodies, which additive helps to prevent aggregation by forming disulfide bonds correctly?

  • High concentration of SDS
  • Redox pair such as reduced and oxidized glutathione (GSH/GSSG)
  • Excess imidazole
  • Ammonium sulfate

Correct Answer: Redox pair such as reduced and oxidized glutathione (GSH/GSSG)

Q16. Which chromatography mode is best suited for purifying antibodies using antigen-specific interactions?

  • Size-exclusion chromatography
  • Hydrophobic interaction chromatography
  • Protein A/G affinity chromatography
  • Ion-exchange chromatography

Correct Answer: Protein A/G affinity chromatography

Q17. In a purification table, what does “specific activity” signify?

  • Total protein mass in the sample
  • Activity per unit protein (e.g., U/mg), indicating purity with respect to function
  • Absolute yield of enzyme in mg
  • Volume of sample required for assay

Correct Answer: Activity per unit protein (e.g., U/mg), indicating purity with respect to function

Q18. Which HPLC mode often denatures proteins due to use of organic solvents and is therefore used mainly for analytical characterization rather than activity-preserving purification?

  • Reversed-phase HPLC
  • Size-exclusion HPLC under aqueous conditions
  • Ion-exchange HPLC
  • Affinity HPLC with mild buffers

Correct Answer: Reversed-phase HPLC

Q19. Which parameter is most indicative of a successful scale-up from lab to pilot-scale protein purification?

  • Identical column dimensions as lab-scale
  • Maintenance of product quality (activity and purity) and acceptable yield/recovery
  • Use of identical volumes of buffers
  • Shorter residence times regardless of flow dynamics

Correct Answer: Maintenance of product quality (activity and purity) and acceptable yield/recovery

Q20. Protease inhibitors are routinely added during cell lysis primarily to prevent which problem?

  • Protein aggregation due to salts
  • Proteolytic degradation of target proteins by endogenous proteases
  • Loss of affinity tag binding
  • Changes in isoelectric point

Correct Answer: Proteolytic degradation of target proteins by endogenous proteases

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