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Chapter 4

Chapter 4. Proteins as Products. Proteins as Biotech Products. Enzymes – speed up chemical reactions Synthesis – combines small molecules to make larger molecules ATP synthetase, peptidyl transferase, polymerase Depolymerization – breaks down large molecules Amylase, lipase, protease

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Chapter 4

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  1. Chapter 4 Proteins as Products

  2. Proteins as Biotech Products • Enzymes – speed up chemical reactions • Synthesis – combines small molecules to make larger molecules • ATP synthetase, peptidyl transferase, polymerase • Depolymerization – breaks down large molecules • Amylase, lipase, protease • Hormones – carry chemical messages • Antibodies – part of immune response

  3. Proteins as Biotech Products • Therapeutic Protein • used to treat a disease that is caused by a gene that fails to produce a necessary protein or that produces a dysfunctional protein

  4. Proteins as Biotech Products • Food processing – the creamy in ice cream • Textile and leather goods – bio-bleaches • Detergents – enzymes to dissolve stains • Paper manufacturing and recycling – reduce negative environmental impacts • Adhesives – barnacles and mussels • Bioremediation – proteins used to clean up harmful waste

  5. Central Dogma • DNA codes for RNA which codes for proteins.

  6. Translation • A protein is a string of amino acids held together by peptide bonds and do most of the work in a cell

  7. Translation

  8. Translation

  9. Protein Structure • Once the amino acid chain is released from the ribosome, a number of modifications are made in order for the protein to perform it’s intended function. • The protein must fold into it’s appropriate 3-dimensional shape.

  10. Protein Structure • Proper folding of the protein is essential for it’s activity because it must bind it’s substrate to perform it’s job.

  11. Protein Structure • Primary – Peptide bonds in a chain of amino acids • Secondary – Hydrogen bonding between amino acids forms alpha-helices and beta-sheets • Tertiary – three dimensional folding of protein due to disulfide linkages and hydrophobic interactions between alpha-helices and beta-sheets • Quaternary – aggregation of multiple polypeptide chains

  12. Protein Structure • Glycosylation • Carbohydrate units added to protein • Increases solubility, orients protein in membrane, extends life of protein • Occurs in the golgi

  13. Protein Structure Coding: Template: mRNA: tRNA: amino acid: 5’-GATCTGAATCGCTATGGC-3’ 3’-CTAGACTTAGCGATACCG-5’ mRNA 5’-GAUCUGAAUCGCUAUGGC-3’ CUAGACUUAGCGAUACCG Asp, Leu, Asn, Arg, Tyr,Gly

  14. Protein Structure • DNA codes for proteins that confer traits

  15. Protein Engineering • Directed Molecular Evolution • Introducing specific, predefined alterations in the DNA sequence.

  16. Protein Production • Steps in bioprocessing

  17. Protein Expression • Bacteria • Advantages • Cheap and easy to grow • Biology is well-defined • High yield of recombinant proteins in culture • Disadvantages • Many proteins become insoluble in inclusion bodies • Most if not all post-translational modifications are not added

  18. Protein Expression • Fungi • Advantages • Grown in simple, inexpensive media • Secrete many proteins into the media • Capable of many post-translational modifications • Disadvantages • Recombinant proteins usually expressed at low levels • Some post-translational modifications differ significantly

  19. Protein Expression • Plants • Advantages • Rapid growth and reproductive rates • Perform most post-translational modifications • Transgenic plants can be self-fertilized • Disadvantages • Not all mammalian proteins are expressed in plants • Plant cells have a tough cell wall • Some plants produce proteins in their green leaf tissues

  20. Protein Expression • Mammalian Cell Culture • Advantages • Protein-folding and post-translational modification • Powerful promoters to regulate protein expression • High expression levels • Disadvantages • Complex and expensive nutritional requirements • Slow growing

  21. Protein Extraction • The target protein must be separated from the complex mixture of biological molecules

  22. Protein Extraction • Isolated proteins must be stabilized • Very sensitive to changes in temperature • Proteases that could digest the target protein are a threat • Protein folding is dependent on the pH of the environment

  23. Protein Purification • Chromatography • A method to separate proteins by size, charge, or chemical properties as they pass through a column of resin beads Chromatography Animation

  24. Protein Purification • Chromatography • Resin/matrix – solid particles in the column • Sample – protein mixture that is loaded on the column • Elution – liquid that passes through the column and is collected in fractions

  25. Protein Purification • Size Exclusion Chromatography • Separates proteins based on size • Small molecules get caught in the beads • Larger molecules pass quickly around the beads and elute first

  26. Protein Purification • Ion Exchange Chromatography • Separates molecules based on ionic charge • Proteins are eluted by increasing the concentration of a salt buffer • Proteins with the weakest charge are eluted first

  27. Protein Purification • Hydrophobic Interaction Chromatography • Separates proteins based on repulsion to water • Proteins are eluted by decreasing the salt concentration of the buffer • The least hydrophobic proteins are eluted first

  28. Protein Purification • Affinity Chromatography • Separates proteins based on molecular conformation • Matrix is made of a ligand specific for the desired protein • The protein is cleaved from the matrix using a site-specific protease

  29. Protein Purification • High Performance Liquid Chromatography (HPLC) • Applies high pressure to drive sample through the column faster

  30. Protein Verification • SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) • Separates proteins in an electrical field based on molecular size

  31. Protein Verification • Sodium Dodecyl Sulfate (SDS) • A detergent that denatures the secondary and tertiary structure of the protein • Coats the protein with negative charges Add SDS

  32. Protein Verification • Polyacrylamide Gel Electrophoresis (PAGE) • Much tighter gel matrix than agarose, which makes polyacrylamide ideal for separating proteins

  33. Protein Verification • SDS-PAGE to test for purity

  34. Protein Verification SDS-PAGE Animation

  35. Application • Recombinant human insulin

  36. Preserving Proteins • Lyophilization (freeze drying) • Placed under vacuum to hasten evaporation of water • Containers are sealed after water is removed

  37. Scale-up of Protein Purification • R&D works on small scale • Large production demands protocols to scale-up bioreactors • If FDA approval has been gained for small-scale, cannot change the parameters when scaled up

  38. Postpurification Analysis • Protein Sequencing • Determining the order of amino acids • X-ray Crystallography • Determining tertiary and quaternary structure of protein

  39. Proteomics • Proteomes are compared under healthy and diseased states • The variations of protein expression are then correlated to onset or progression of a specific disease • Protein Microarrays • Identifies protein interactions

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