N-terminal and C-terminal tags MCQs With Answer

N-terminal and C-terminal tags MCQs With Answer

This quiz set focuses on N‑terminal and C‑terminal affinity and detection tags commonly used in protein expression, purification, and characterization—tailored for M.Pharm students studying Proteins and Protein Formulations. Questions cover tag types (His, GST, MBP, FLAG, Strep, SUMO), choice of tag position, effects on protein solubility, activity and structure, protease cleavage strategies, linker design, host expression considerations, and downstream processing implications such as elution conditions and immunogenicity. Emphasis is placed on practical decision-making for designing constructs and interpreting experimental outcomes. These MCQs are intended to deepen conceptual understanding and prepare students for laboratory design and troubleshooting when working with tagged recombinant proteins.

Q1. Which of the following is the primary reason to choose an N‑terminal tag over a C‑terminal tag for a recombinant protein?

• Because N‑terminal tags are always smaller than C‑terminal tags
• To avoid interfering with a C‑terminal signal sequence or functional domain
• Because C‑terminal tags cannot be cleaved by proteases
• Because N‑terminal tags increase protein pI more than C‑terminal tags

Correct Answer: To avoid interfering with a C‑terminal signal sequence or functional domain

Q2. Which affinity tag is most commonly used with immobilized metal affinity chromatography (IMAC)?

• GST
• His‑tag (polyhistidine)
• MBP
• FLAG

Correct Answer: His‑tag (polyhistidine)

Q3. Why are flexible linkers (e.g., Gly‑Ser repeats) often inserted between a tag and the target protein?

• To increase the molecular weight for easier detection on SDS‑PAGE
• To provide protease recognition sites for tag removal
• To reduce steric hindrance and allow proper folding and efficient protease access
• To fix the tag in a rigid orientation for crystallography

Correct Answer: To reduce steric hindrance and allow proper folding and efficient protease access

Q4. Which protease is widely used for precise tag removal because it leaves only a single glycine residue in many constructs and has a well‑defined recognition site?

• Thrombin
• Factor Xa
• TEV protease
• Enterokinase

Correct Answer: TEV protease

Q5. When expressing a secreted protein with a signal peptide, where should an affinity tag typically be placed to avoid disrupting secretion?

• Immediately upstream of the signal peptide
• At the N‑terminus before the signal peptide
• C‑terminal to the mature protein (after signal peptide cleavage site)
• Within the signal peptide sequence

Correct Answer: C‑terminal to the mature protein (after signal peptide cleavage site)

Q6. Which tag is known both for improving solubility of difficult proteins and for enabling amylose affinity purification?

• GST
• MBP (maltose‑binding protein)
• His‑tag
• FLAG

Correct Answer: MBP (maltose‑binding protein)

Q7. What is a common disadvantage of leaving a large fusion tag (e.g., GST or MBP) uncleaved for downstream structural studies?

• It always increases thermal stability making folding studies impossible
• It can mask functional sites, alter folding, and impede crystallization or NMR analysis
• It prevents purification by affinity chromatography
• Uncleaved large tags are invisible in mass spectrometry

Correct Answer: It can mask functional sites, alter folding, and impede crystallization or NMR analysis

Q8. Which detection tag is a short hydrophilic epitope commonly used for antibody‑based detection and immunoprecipitation?

• Strep‑tag II
• Polyhistidine tag
• FLAG tag
• MBP tag

Correct Answer: FLAG tag

Q9. When planning to remove a tag by protease cleavage, which factor most strongly influences cleavage efficiency?

• The color of the purification column
• Accessibility and local structure at the cleavage site
• Length of the tag only
• Whether the tag is at the N‑terminus or C‑terminus solely

Correct Answer: Accessibility and local structure at the cleavage site

Q10. Which tag system allows gentle elution under native conditions by competition with biotin analogs and is useful for preserving complex integrity?

• GST on glutathione resin
• His‑tag on Ni‑NTA with high imidazole
• Streptavidin‑biotin (desthiobiotin elution) / Strep‑tag system
• FLAG peptide elution with SDS

Correct Answer: Streptavidin‑biotin (desthiobiotin elution) / Strep‑tag system

Q11. Which consideration is most important when choosing a tag for expression in E. coli to minimize inclusion body formation?

• Select the shortest possible tag regardless of function
• Choose solubility‑enhancing tags (e.g., MBP, NusA) and optimize expression conditions
• Always use C‑terminal tags in E. coli
• Use stronger promoters to increase expression yield

Correct Answer: Choose solubility‑enhancing tags (e.g., MBP, NusA) and optimize expression conditions

Q12. Which tag is most appropriate if downstream application requires minimal residual non‑native residues after cleavage?

• Tags cleaved by TEV protease with optimized junctions
• Large tags like GST left uncleaved
• Any tag followed by random protease
• Tags with long rigid linkers that cannot be cleaved

Correct Answer: Tags cleaved by TEV protease with optimized junctions

Q13. How can an N‑terminal His‑tag affect a protein’s measured isoelectric point (pI)?

• It always lowers pI dramatically by introducing negative charges
• It can raise or slightly alter the pI by adding positively charged histidines, affecting solubility and purification behavior
• It has no effect because tags are ignored in pI calculations
• It converts the protein into a membrane protein

Correct Answer: It can raise or slightly alter the pI by adding positively charged histidines, affecting solubility and purification behavior

Q14. Which method is recommended to confirm complete removal of an affinity tag after protease treatment?

• Only check absorbance at 280 nm
• Analyze by SDS‑PAGE and mass spectrometry to verify size change and mass accuracy
• Assume cleavage based on incubation time
• Rely solely on Western blot against the target protein

Correct Answer: Analyze by SDS‑PAGE and mass spectrometry to verify size change and mass accuracy

Q15. Why might a C‑terminal tag be preferred when the N‑terminus undergoes co‑translational modifications (e.g., N‑terminal acetylation) that are functionally important?

• Because C‑terminal tags are cheaper to synthesize
• To preserve native N‑terminal modifications and function while facilitating purification/detection at the C‑terminus
• Because proteases cannot remove N‑terminal tags
• To make the protein fluorescent

Correct Answer: To preserve native N‑terminal modifications and function while facilitating purification/detection at the C‑terminus

Q16. Which tag is typically used for purification with glutathione resin and often forms dimers that may influence oligomeric state?

• His‑tag
• GST
• Strep‑tag
• FLAG tag

Correct Answer: GST

Q17. In designing constructs for eukaryotic expression, what additional element should be considered together with N‑terminal tagging to ensure efficient translation initiation?

• Including a bacterial Shine‑Dalgarno sequence
• Ensuring an appropriate Kozak consensus sequence around the start codon
• Always using a TEV site immediately after the start codon
• Placing the tag inside an intron

Correct Answer: Ensuring an appropriate Kozak consensus sequence around the start codon

Q18. Which approach reduces the risk that affinity purification will co‑purify tag partners or contaminants binding to the tag rather than the target protein?

• Using two different orthogonal tags (tandem affinity purification, TAP) and sequential affinity steps
• Using only a very long single tag
• Purifying at extremely high temperatures
• Using no washing steps during affinity purification

Correct Answer: Using two different orthogonal tags (tandem affinity purification, TAP) and sequential affinity steps

Q19. Which statement best describes how tag location can influence protein crystallization prospects?

• Tag location never affects crystallization because tags are flexible
• Tags at termini can increase disorder; removing or relocating the tag can reduce heterogeneity and improve crystal packing
• Only internal tags influence crystallization
• Adding multiple tags always improves crystallization by increasing solubility

Correct Answer: Tags at termini can increase disorder; removing or relocating the tag can reduce heterogeneity and improve crystal packing

Q20. Which consideration is most relevant when selecting a tag for therapeutic protein production regarding regulatory and immunogenicity concerns?

• Tags are irrelevant for regulatory submissions
• Choose tags and cleavage strategies that minimize residual non‑native sequences and immunogenic epitopes in the final product
• Always leave tags on to improve stability even in the final therapeutic formulation
• Prefer tags that add glycosylation sites regardless of function

Correct Answer: Choose tags and cleavage strategies that minimize residual non‑native sequences and immunogenic epitopes in the final product

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