1. Protein Pharmaceuticals (I) “Isolation, Synthesis, and Recombination”*
07/16/96
Protein Pharmaceuticals (I) “Isolation, Synthesis, and Recombination”
Dr. Aws Alshamsan
Department of Pharmaceutics
Office: AA87
Tel: *

2. Objectives of this lecture
By the end of this lecture you will be able to:
Understand the role of proteins in health and disease
Recognize the limitations of traditional protein production methods
Recognize the advantages of recombinant over naturally-isolated proteins

3. Our genetic material speaks the tale of proteins
Proteins will provide almost all structural and functional aspects of the living organism

4. reverse transcriptase
Functions of Proteins
Structural
Enzymes
DNA Regulation
Immune
antibodies
DNA polymerase III
collagen
luciferase
amylase
reverse transcriptase

5. Disease-causing proteins
Proteins in diseases and therapy
Disease-causing proteins

6. Loss in protein production or functionality might lead to serious diseases
Diabetes Mellitus
Sickle cell anemia

7. C-reactive protein Proteins might mediates diseases as well
C-reactive protein mediates cardiovascular disorders
C-reactive protein

8. Exogenous proteins might mediate serious reactions
Hypersensitivity reactions

9. Proteins in diseases and therapy
Therapeutic Proteins

10. Serving or mimicking an endogenous function
Hormonal therapy (Insulin, GH)

11. Exploiting an endogenous reaction
Vaccines

12. Antagonizing an endogenous function*
07/16/96
Antagonizing an endogenous function
mAb therapy (Herceptin) *

14. * 07/16/96 * Protein Pharmaceuticals Polypeptides and Proteins
Natural extraction
DNA recombination
Oligopeptides
Chemical synthesis *

15. Protein Pharmaceuticals
Peptide: consists of 2 or more amino acids
Oligopeptide: consists of 10 or fewer amino acids
Polypeptides: chains of 10 to 50 amino acids
Proteins: polypeptides of more than 50 amino acids

16. Naturally-Occurring Proteins Site of action includes
Hormone
Size (a.a.)
Location
Site of action includes
ACTH 39
Anterior pituitary
Adrenal cortex
ADH (VP) 9
Posterior pituitary
Kidny / CVS / CNS
Calcitonin 32
Thyroid
Bones GH
191
Bones / muscles / liver
Oxytocin
Mammary gld. / Uterus / CNS
Prolactin
198
Mammary gland
Insulin 51
Pancreas
Liver / Muscles / Adipocytes

17. Oligopeptides
Many hormones in our body are actually oligopeptides such as Oxytocin and Vasopressin (ADH)

18. Oligopeptides
Pharmaceutical production of oligopeptides can be achieved by:
Extraction from natural sources (e.g. salmon calcitonin)
Solid-phase peptide synthesis (SPPS)
Recombinant DNA technology

19. Solid Phase Peptide Synthesis
Method developed by Bruce Merrifield and published in JACS in 1963
He won the Nobel Prize in Chemistry in 1984
Bruce Merrifield

20. Solid Phase Peptide Synthesis

21. Solid Phase Peptide Synthesis

22. Solid Phase Peptide Synthesis
Very efficient synthesis (>99% per couple)
10 residue peptide, 99% coupling
Yield = = 90.4%
50 residue peptide, 99% coupling
Yield = = 60.5%
Limited to small peptides
No glycosylation ability

23. Polypeptides and Proteins
Other hormones in our body consist of larger peptide chains or more complex proteins such as GH, ACTH, and Prolactin GH

24. Blood Fractionation Cells Blood Plasma Plasma Proteins Thrombocytes
Leukocytes
Cells
Erythrocytes

25. Cohn Process
Edwin Cohn

26. Blood Fractionation

27. Polypeptides and Proteins
Natural sources are often rare and expensive
Difficult to keep up with demand
Hard to isolate product
Lead to immune reactions (e.g. HAMA response)
Viral & pathogen contamination

28. Polypeptides and Proteins
Most protein pharmaceuticals today are recombinant products
Cheaper, safer, abundant supply

29. Six-Step Process Isolation of gene of interest
Introduction of gene to expression vector
Transformation into host cells
Growth of cells through fermentation
Isolation & purification of protein
Formulation of protein product

30. Requirements for recombinant protein production
Vector
Gene
Host
Plasmid
Insulin
E. coli

31. Vector pUC18 Selectable marker
a gene (antibiotic resistance) when expressed on plasmid will allow host cells to survive
Polylinker (multiple cloning site MSC) is a short segment of DNA which contains many (up to ~20) restriction sites
Promoter is a short DNA sequence which enhances expression of adjacent gene
Ori origin of replication is a particular sequence where replication is initiated

32. Restriction Nucleases
Enzymes that cut double-stranded DNA at specific recognition nucleotide sequences known as restriction sites (palindrome)
مودته تدوم لكل هول *** وهل كل مودته تدوم

33. Ligases
DNA ligation is the act of joining together DNA strands with covalent bonds with the aim of making new viable DNA or plasmid
T4 DNA ligase has the unique ability to join sticky and blunt ended fragments

34. DNA Recombination

35. Choice of production vehicle
In principle, any protein can be produced using any genetically engineered organism
Bacteria
Yeast
Mammalian

36. Now you are able to:
Understand the role of proteins in health and disease
Recognize the limitations of traditional protein production methods
Recognize the advantages of recombinant over naturally-isolated protein

37. Next Lecture
Recombination and production of protein pharmaceuticals