Production of insulin through recombinant DNA technology is a core topic for B. Pharm students, integrating molecular cloning, protein expression, and bioprocessing. This overview emphasizes essential keywords: insulin gene cloning, expression vectors, E. coli and yeast hosts, codon optimization, inclusion bodies, disulfide bond formation, proinsulin processing, affinity purification, refolding, downstream processing, endotoxin removal, analytical QC (SDS-PAGE, HPLC, bioassays), scale-up, and biosimilar regulation. Understanding these concepts prepares students for practical challenges in pharmaceutical manufacturing and regulatory compliance. Clear knowledge of molecular and process steps is crucial for designing safe, effective recombinant insulin products. ‘Now let’s test your knowledge with 30 MCQs on this topic.’
Q1. Which host organism is most commonly used for high-yield recombinant human insulin production in early industrial processes?
- Escherichia coli
- Saccharomyces cerevisiae
- Pichia pastoris
- Chinese hamster ovary (CHO) cells
Correct Answer: Escherichia coli
Q2. What is the primary reason E. coli often produces insulin as inclusion bodies?
- Excessive glycosylation in the cytoplasm
- Rapid overexpression leading to misfolding and aggregation
- Strong periplasmic secretion signals
- High protease activity preventing aggregation
Correct Answer: Rapid overexpression leading to misfolding and aggregation
Q3. In recombinant insulin production, what is the typical strategy to obtain correctly paired disulfide bonds?
- Secretion into the reducing cytoplasm
- In vitro oxidative refolding with redox shuffling agents
- Maintaining anaerobic fermentation conditions
- Using prokaryotic glycosylation systems
Correct Answer: In vitro oxidative refolding with redox shuffling agents
Q4. Which molecular construct is often expressed to simplify insulin processing and yield proper chain formation?
- Separate A and B chains cloned on different plasmids
- Preproinsulin with signal peptide retained
- Proinsulin (single-chain) that is enzymatically cleaved to mature insulin
- Fusion to large carrier proteins without cleavage sites
Correct Answer: Proinsulin (single-chain) that is enzymatically cleaved to mature insulin
Q5. Which enzyme combination is classically used to convert proinsulin to mature insulin chains after expression?
- Trypsin and carboxypeptidase B
- Pepsin and chymotrypsin
- DNase I and RNase A
- Lysozyme and protease K
Correct Answer: Trypsin and carboxypeptidase B
Q6. Why is codon optimization performed when expressing human insulin in E. coli?
- To introduce N-linked glycosylation sites
- To match host tRNA abundance and improve translation efficiency
- To add signal peptides for secretion into the extracellular medium
- To convert disulfide bonds into thioether bonds
Correct Answer: To match host tRNA abundance and improve translation efficiency
Q7. Which plasmid feature determines how many copies of the insulin expression vector exist per bacterial cell?
- Promoter sequence
- Origin of replication (ori)
- Multiple cloning site (MCS)
- Terminator sequence
Correct Answer: Origin of replication (ori)
Q8. What is the role of an affinity tag, such as a His-tag, in recombinant insulin production?
- To enhance disulfide bond formation
- To facilitate purification via metal affinity chromatography
- To enable secretion into the periplasm
- To increase glycosylation efficiency
Correct Answer: To facilitate purification via metal affinity chromatography
Q9. Which promoter is commonly used for high-level, inducible expression in BL21(DE3) strains?
- Lac promoter without T7 polymerase
- T7 promoter recognized by T7 RNA polymerase
- GAL1 promoter from yeast
- CMV promoter for mammalian expression
Correct Answer: T7 promoter recognized by T7 RNA polymerase
Q10. What is the primary challenge when expressing insulin in E. coli compared with yeast?
- Excessive N-linked glycosylation in E. coli
- Lack of eukaryotic post-translational modifications and oxidative environment for disulfide formation in E. coli cytoplasm
- Lower growth rate of E. coli than yeast
- Difficulty in plasmid transformation of E. coli
Correct Answer: Lack of eukaryotic post-translational modifications and oxidative environment for disulfide formation in E. coli cytoplasm
Q11. Which downstream step specifically removes bacterial endotoxins important for injectable insulin products?
- Affinity chromatography with His-tag
- Anion-exchange chromatography and ultrafiltration for endotoxin clearance
Correct Answer: Anion-exchange chromatography and ultrafiltration for endotoxin clearance
Q12. During refolding of insulin from inclusion bodies, which pair of reagents is commonly used to create a redox environment for correct disulfide pairing?
- Dithiothreitol (DTT) only
- Oxidized and reduced glutathione (GSSG/GSH)
- SDS and urea
- EDTA and EGTA
Correct Answer: Oxidized and reduced glutathione (GSSG/GSH)
Q13. What analytical technique is used to confirm the molecular mass and correct chain formation of recombinant insulin?
- Northern blotting
- MALDI-TOF or ESI mass spectrometry
- Gram staining
- Plate count assay
Correct Answer: MALDI-TOF or ESI mass spectrometry
Q14. Why is periplasmic expression of proinsulin in E. coli sometimes preferred?
- Periplasm is more reducing than cytoplasm promoting aggregation
- Periplasmic space supports disulfide bond formation and simplifies purification
- It increases plasmid copy number
- It enables glycosylation of insulin
Correct Answer: Periplasmic space supports disulfide bond formation and simplifies purification
Q15. Which fermentation mode is often used industrially to maximize recombinant protein yield, including insulin precursors?
- Continuous culture with constant dilution
- Batch culture without feeding
- Fed-batch fermentation to control nutrient supply and reduce acetate formation
- Solid-state fermentation
Correct Answer: Fed-batch fermentation to control nutrient supply and reduce acetate formation
Q16. What is the function of a signal peptide when engineering yeast to secrete proinsulin?
- Promote cytoplasmic aggregation
- Direct the nascent protein into the secretory pathway for extracellular secretion
- Increase plasmid stability
- Prevent disulfide bond formation
Correct Answer: Direct the nascent protein into the secretory pathway for extracellular secretion
Q17. In the context of regulatory approval, what distinguishes a biosimilar insulin from a generic small-molecule drug?
- Biosimilars are chemically identical to innovators
- Biologics are complex proteins requiring demonstration of similarity in structure, function, and clinical performance rather than identical composition
- Biosimilars do not require manufacturing controls
- Generics require clinical trials but biosimilars do not
Correct Answer: Biologics are complex proteins requiring demonstration of similarity in structure, function, and clinical performance rather than identical composition
Q18. Which parameter is most critical to control during induction of protein expression with IPTG?
- IPTG concentration, induction temperature, and induction time to balance yield and solubility
- Final pH only
- Antifoam addition frequency
- Optical rotation of the culture
Correct Answer: IPTG concentration, induction temperature, and induction time to balance yield and solubility
Q19. What is the role of SDS-PAGE in insulin production QC?
- Quantify endotoxin levels
- Assess purity, molecular weight and detect degradation products
- Measure disulfide bond formation directly
- Determine glycosylation pattern
Correct Answer: Assess purity, molecular weight and detect degradation products
Q20. Which strategy can reduce formation of inclusion bodies during recombinant insulin expression?
- Increase induction temperature to 42°C
- Lower induction temperature and reduce induction strength
- Use a stronger promoter and higher copy plasmid
- Express in stationary phase only
Correct Answer: Lower induction temperature and reduce induction strength
Q21. Why is enzymatic cleavage of fusion partners often necessary in insulin production?
- Fusion partners always improve biological activity
- Fusion facilitates expression/purification but must be removed to yield native insulin sequence and activity
- Fusion partners are required to form disulfide bonds
- To glycosylate insulin properly
Correct Answer: Fusion facilitates expression/purification but must be removed to yield native insulin sequence and activity
Q22. What is a common method to solubilize inclusion bodies before refolding?
- High-salt buffer without denaturant
- Strong chaotropes like 6–8 M urea or 6 M guanidine hydrochloride
- Cold water wash only
- Treatment with proteases
Correct Answer: Strong chaotropes like 6–8 M urea or 6 M guanidine hydrochloride
Q23. Which property makes Pichia pastoris an attractive host for recombinant insulin analogs?
- Lacks secretory pathway
- High-density growth, strong inducible promoters (AOX1), and ability to perform some eukaryotic folding
- Prokaryotic transcription machinery
- Intrinsic production of human-like glycosylation
Correct Answer: High-density growth, strong inducible promoters (AOX1), and ability to perform some eukaryotic folding
Q24. Which analytical assay directly measures biological potency of recombinant insulin?
- Endotoxin assay
- Cell-based receptor binding or glucose uptake bioassay
- UV absorbance at 280 nm only
- Gram staining of the product
Correct Answer: Cell-based receptor binding or glucose uptake bioassay
Q25. During scale-up, which variable is commonly optimized to prevent oxygen limitation affecting protein expression?
- pH setpoint only
- Aeration and agitation to maintain dissolved oxygen levels
- Plasmid sequence
- Type of SDS used in downstream processing
Correct Answer: Aeration and agitation to maintain dissolved oxygen levels
Q26. What is the primary reason to perform codon harmonization instead of simple codon optimization?
- To increase glycosylation sites
- To preserve translation kinetics and co-translational folding while adapting to host usage
- To remove all rare codons completely
- To insert protease cleavage sites indiscriminately
Correct Answer: To preserve translation kinetics and co-translational folding while adapting to host usage
Q27. Which safety concern must be addressed specifically when producing insulin in bacterial hosts?
- Viral contamination from mammalian serum
- Bacterial endotoxin contamination that can cause pyrogenic reactions
- Excessive glycosylation leading to immunogenicity
- Radioactive contamination
Correct Answer: Bacterial endotoxin contamination that can cause pyrogenic reactions
Q28. What is the advantage of using recombinant DNA-derived insulin over animal-derived insulin?
- Higher antigenicity and more allergic reactions
- Identical sequence to human insulin, reduced immunogenicity, and scalable consistent manufacturing
- Lower purity overall
- Inability to modify pharmacokinetics
Correct Answer: Identical sequence to human insulin, reduced immunogenicity, and scalable consistent manufacturing
Q29. Which chromatographic method is most selective for purification of His-tagged insulin precursors?
- Size-exclusion chromatography only
- Immobilized metal affinity chromatography (IMAC)
- Reverse-phase HPLC without prior desalting
- Simple ethanol precipitation
Correct Answer: Immobilized metal affinity chromatography (IMAC)
Q30. Which regulatory documentation is essential to demonstrate consistent manufacturing and control of recombinant insulin?
- Certificate of analysis, batch records, process validation reports, and comparability data for biosimilars
- Only a simple product label
- Only academic publications about the gene sequence
- Oral inspection reports without documentation
Correct Answer: Certificate of analysis, batch records, process validation reports, and comparability data for biosimilars
I am a Registered Pharmacist under the Pharmacy Act, 1948, and the founder of PharmacyFreak.com. I hold a Bachelor of Pharmacy degree from Rungta College of Pharmaceutical Science and Research. With a strong academic foundation and practical knowledge, I am committed to providing accurate, easy-to-understand content to support pharmacy students and professionals. My aim is to make complex pharmaceutical concepts accessible and useful for real-world application.
Mail- Sachin@pharmacyfreak.com