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Protein (Bio)Technology; Industrial and Medical Perspectives

Protein (Bio)Technology; Industrial and Medical Perspectives. M. Kamran Azim, Ph.D. International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry, Dr. Panjwani Center for Molecular Medicine and Drug Research University of Karachi.

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Protein (Bio)Technology; Industrial and Medical Perspectives

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  1. Protein (Bio)Technology; Industrial and Medical Perspectives M. Kamran Azim, Ph.D. International Center for Chemical and Biological Sciences H.E.J. Research Institute of Chemistry, Dr. Panjwani Center for Molecular Medicine and Drug Research University of Karachi

  2. Central dogma of molecular biology

  3. DNA for InformationProtein for Execution

  4. Proteins and the molecular basis of life • Proteins have dynamic and diverse role(s) in the body • Catalysing biochemical reactions (Enzymes; Regulators) • Formimg receptors and channels in cell membranes (Membrane proteins) • Providing intra/extracellular scaffolding support (Cytoskeleton protein) • Transporting molecules (Transport proteins) • Hormones • more

  5. The central role of proteome

  6. R1 R2 C C +NH3 +NH3 CO2- CO2- H H The Basics - Proteins Polymers of amino acids (20 naturally occurring) R1 H H2O + H C N CO2- +NH3 C C H O R2 R1 = CH3 Alanine Ala A R2 = CH3OH Serine Ser S AlanylSerine Ala-Ser AS

  7. primary structure – amino acid sequence secondary structure – local spatial arrangement DRLEFIVTALLKPW N-terminus C-terminus a-helix b-sheet http://mcl1.ncifcrf.gov/integrase/asv_secstr.html tertiary structure – protein folding quaternary structure – multimeric complexes http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html The Basics – Protein Structure

  8. Proteins in Biotechnology Industrial and Medical Applications

  9. Enzymes having important industrial applications

  10. Protein Therapeutics

  11. Protein Therapeutics (PTs) • Protein and peptides in clinical use • In recent years, Protein therapeutics have increased dramatically • First recombinant protein therapeutic-human insulin, introduced in 1982 • >125 Protein therapeutics currently in use • Several advantages over small-molecule drugs. • Proteins serve a highly specific and complex set of functions • No interference with normal biological processesNo side effects • Well toleratedNo elicit immune responses • Development and FDA approval is faster • Due to unique form-function of proteinFar-reaching patent protection

  12. Protein Therapeutics (PTs) • Relatively small number of PTs are purified from native sources • Pancreatic enzymes bovine or porcine sources • Alpha-1-proteinase inhibitor from human plasma • Most PTs are produced by recombinant DNA technology and purified from a wide range of organisms. • Production systems for recombinant proteins include bacteria, yeast, insect cells, mammalian cells and transgenic animals and plants. • The system of choice depends • on the cost of production • Protein modification (e.g. glycosylation, phosphorylation etc.)

  13. Human Insulin in the treatment of Diabetes mellitus • Diabetes mellitus is common metabolic disease characterized by hyperglycaemia due to lack of the protein hormone Insulin. • Insulin signals cells to perform numerous functions related to glucose hemeostasis and intermediary metabolism • 1922 • Insulin was first purified from bovine and porcine pancreas and used as daily injection • Problems: the availability of animal pancreas; cost of insulin purification; immunological reactions. • 1982 • Solution: Isolation of human insulin gene; Engineering of E. coli to express human insulin by rDNA technology, growing vast quantities of bacteria, large-scale production of human insulin • Result: Abundant and inexpensive human insulin with low immunogenicity and free from animal pancreatic substances.

  14. Categorization of FDA approved PTs based on mechanism of action • PTs replacing a protein that is deficient/adnormal • PTs augmenting an existing pathway • PTs providing a novel function • PTs that interfere with a molecule/organism • PTs that deliver other compounds/proteins • Protein vaccines • Protein diagnostics

  15. PTs replacing a protein that is deficient/adnormal of actionSpecific endogenous protein is deficientRemediation by exogenous protein

  16. PTs augmenting an existing pathwayTo enhance the magnitude/timing of a particular normal protein activity

  17. PTs providing a novel functionForeign proteins with novel activity

  18. PTs that interfere with a molecule/organismMonoclonal Antibodies (mAb) guide the immune system to destroy targeted molecules/cells

  19. PTs that deliver other compounds/proteinsFor selective delivery of small-molecule and protein to the target

  20. Protein vaccines Prophylactic and therapeutic vaccines

  21. Protein diagnostics Recombinant proteins for medical diagnostics

  22. Techniques in Protein technology research • How PTs can be produced? • Necessary theoretical knowledge. • What types of skills required?

  23. Necessary theoretical knowledge. • Molecular and Cellular Biology Comprehensive understanding of biochemical processes in health and disease including Signal Transduction etc. Genetic manupulation • Protein (Bio)Chemistry Protein forms and function Protein purification methodology • Fermentation technology

  24. What types of skills required? • Nucleic acid isolation and characterization • Molecular cloning • Protein Over-Expression in a variety of hosts • Protein chromatography using Gel filtration, Ion-exchange, Affinity chromatography • Protein electrophoresis

  25. Flow chart of methods in Protein Technology

  26. Methods in Protein Chromatography • 1) Size • gel filtration/size exclusion • 2) Charge • ion-exchange • cation (basic proteins) and anion (acidic proteins) • 3) Hydrophobic/polar • reversed phase, hydrophobic & hydrophilic interaction

  27. Affinity Chromatography purification • specific interaction with a ligand bound to column • - “general” ligand e.g. chemical group • - immobilised metal -Histidine containing proteins • highly specific ligand • e.g.antibody

  28. Gel Electrophoresis various lengths linear / gradient reducing / non-reducing Multi-gel runners = higher reproducibility

  29. Protein detection and image capture pre-gel sample labelling 35S-methionine Cy3, Cy5, Cy2 (DiGE) post-gel staining colloidal coomassie blue silver SYPRO ruby Pro-Q Diamond – phosphoproteins Pro-Q Emerald – glycoproteins

  30. Stain Examples Coomassie Silver Stain Copper Stain

  31. Flow chart of methods in Protein Technology

  32. Gene Cloning into expression vector (pQE series)

  33. Transformation and Expression

  34. Affinity-tag and desalting purification

  35. Histidine-Tag (metal chelating) affinity chromatography

  36. Histidine-tag (metal chelating) affinity chromatography

  37. Purification to homogeneity checked by Electrophoresis

  38. Intricacy in the ProteinsandFish Harbor, Karachi

  39. Shahjahan masjid in Thatha

  40. Chukundi tomd near Karachi

  41. International Center for Chemical and Biological SciencesH.E.J. Research Institute of ChemistryDr. Panjwani Center for Molecular Medicine and Drug ResearchUniversity of Karachi

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