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Proteins: Building Blocks of Biotech Industries

Discover the role of proteins in biotech products and processes, from enzymes to therapeutic proteins. Explore protein structure, expression, and purification methods. Learn about protein engineering and the impact of DNA codes on protein traits. Understand the significance of protein folding and post-translational modifications in bioprocessing.

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Proteins: Building Blocks of Biotech Industries

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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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