Lecture 01, 2

• What percentage of new drugs are biopharmaceuticals?

  50%
• Why are biopharmaceuticals considered the most profitable?

  They have high market demand and effectiveness
• What are biopharmaceuticals primarily composed of?

  Therapeutic proteins, peptides, or nucleic acids
• What is a heterologous cell system?

  It expresses genes from one organism in another
• What is the purpose of mRNA vaccines like those for Covid-19?

  To provide immunity against the virus
• What are monoclonal antibodies used for?

  Targeted therapy in various diseases
• What is the role of a vector in protein expression?

  To carry the gene for protein production
• What factors should be considered when choosing a promoter for gene expression?

  Type, efficiency, and inducibility
• What is the significance of plasmid stability in dividing cells?

  It affects the consistency of protein production
• What does YIp stand for?

  Yeast integrated plasmid
• What are the components of a plasmid re-engineered for heterologous protein production?

  • Selection marker (e.g., antibiotic resistance)
  • CEN sequence (for fungi)
  • Origin of replication (ORI)
  • Heterologous gene
  • Purification tag
  • Promoter sequence
  • Signal sequence (if secretion is needed)
• Why is high-level expression of proteins important?

  It maximises yield for therapeutic use
• What are inclusion bodies in protein production?

  Insoluble complexes of protein and RNA
• Why is correct disulfide folding important in proteins?

  It ensures proper protein structure and function
• What is the role of Protein Disulfide Isomerase (PDI)?

  It assists in correct disulfide bond formation
• How can fusion proteins help in protein production?

  They facilitate proper folding and stability
• What can cause degradation of recombinant proteins?

  Peptidases or proteases in the cell
• Why is it important to develop strains deficient in specific proteases?

  To prevent degradation of the target protein
• What is metal affinity chromatography used for?

  Purification of therapeutic proteins
• How does histidine tagging work in purification?

  It binds to nickel ions in the column
• What is codon bias in protein expression?

  Preference for specific codons in different organisms
• How can tRNA gene copy number affect protein expression?

  It can increase availability of low-use codons
• What is the Shine-Dalgarno sequence?

  Ribosomal binding site in mRNA
• What issues arise from glycosylation in heterologous proteins?

  Variability in glycosylation across species
• How can glycosylation issues be overcome in protein production?

  By engineering out glycosylation sequences
• What is the purpose of multi-parental DNA shuffling?

  To create variants for improved protein function
• How does artificial evolution improve biopharmaceuticals?

  By selecting variants with enhanced receptor binding
• What are the key features of reengineering a gene for expression in different hosts?

  • Nucleic acid sequence and codon bias
  • Issues with purification (His tagging, affinity domains)
  • Secretion and disulfide bond formation
  • Choice of organism strain for expression
  • Considerations for glycosylation
  • Methods for improving biopharmaceuticals (Artificial Evolution/DNA Shuffling)