Introduction to Proteins as Products

1. Introduction to Proteins as Products Biotechnology 2

2. Review of the Basics • Made up of amino acids • Functions: • Regulatory role • Structural support • Transport • There are literally thousands of functions and we do not yet understand all of them! • In order to understand their functions we have to understand their structure

3. Protein Structure • Polymers of small units (amino acids) • Proteins do NOT have a uniform structure • Due to 20 different amino acids available • The chemical and physical properties are different among the different amino acids • Protein sequence reported by Frederick Sanger in 1953. • Protein folding determines structure

4. Turning Proteins into Products • Identifying proteins and their function is only half the battle. • Once identified, proteins typically need to be grown and then purified and processed into usable, salable products. • Levels of Product Purity (least to most pure) • Research grade • Diagnostic grade • Pharmaceutical grade (low to high dose)

5. Examples of Purified Proteins • Enzymes • Amylases, proteases, lipases (google the company, Genzyme-how many of the enzymes does this company make?)http://www.genzyme.com/business/biz_home.asp • http://www.genzymediagnostics.com/ • Hormones • Antibodies • What was the first recombinant protein to be mass produced? • Cerezyme

6. Basic components of a fermenter

7. Basic steps in bioprocessing Lyophilization

8. More Examples of products: • Food Processing (the creamy in ice cream) • Textiles and Leather Goods (bio-stoning) • Detergents (enzymes) • Paper Manufacturing and Recycling • Adhesives: Natural Glues • Bioremediation: Treating Pollution with Proteins (metallothioneins)

9. Process for making cheese

10. Protein Structures • Levels of Organization • Primary (the AA sequence of its polypeptide chain) • Secondary (H bonding between peptide bonds) • Tertiary (covalent, ionic, H bonding, hydrophobic) • Quaternary (involves more than one subunit)

12. Protein Production Upstream Processing: the actual expression of the protein in the cell • Microorganisms- cheap, well understood, grow rapidly, produce large amounts, clone in as cDNA, fusion gene (fusion protein), inclusion bodies, no glycosylation • Fungi – can do some posttranslational modifications • Plants- 85% of current drugs from plants; rapid growth, cheap, proteins not expressed properly • Mammalian Cell Systems – finicky, grow slowly, and expensive, BUT processes human proteins correctly • Whole-animal –transgenic (goats making spider silk) • Insect systems – baculoviruses are used as vector to insert genes into insect cells

13. Downstream Processing: the protein is separated from other parts of the cell and then isolated from other proteins • Preparing the Protein Extract for Purification • If intracellular, lyse the cells • Detergents or organic solvent can be used for lipid membrane bound proteins • Stabilizing the Proteins in Solution • Temperature, decrease protease activity and denaturing activity, maintain biological activity • Separating the Components in the Extract • Utilize the chemical and physical properties of proteins to separate them

14. Stabilizing the Protein • pH: extremes will denature the protein • Temperature: thermal stability varies among proteins • Typically high temp more damaging • A lot of protein purification happens at 0C or refrigerated conditions • Proteases and nucleases: degradative enzymes • Adsorption surfaces: many proteins denatured by contact w/air, water, glass, or plastic

15. Protein precipitation • Ammonium sulfate • Centrifugation (sized based) • Filtration • Membrane, microfiltration, ultrafiltration • Diafiltration and dialysis • Chromatography • Size-exclusion, ion-exchange, affinity, hydrophobic, iso-electric focusing, 2D electrophoresis • Analytic Methods • HPLC, mass spectrometry

16. Centrifugation

17. Filtration

18. Chromatography Yield: % recovered from final product

19. Hydrophobic chromatography

20. Types of Chromatography • Ion Exchange: Charged molecules bind to oppositely charged group that been immobilized on the matrix • Hydrophobic Interaction Chromatography: non polar groups on the surface of proteins “interact” with the hydrophobic groups. Hydrophobic materials stick tightly together under high salt conditions

21. Hydrophobic interaction chromatography • In the Bio-Rad HIC kit: • Equilibration buffer prepares the column (2M ammonium sulfate buffer) • Elution buffers are low salt concentration (10mM Tris buffer) • Binding buffer: 4 M Ammonium sulfate buffer • Buffers from high salt to low salt concentration • Binding, Equilibration, Wash, Elution buffer

22. Types of Chromatography • Affinity Chromatography: when an impure protein solution is passed through this chromatographic material, the desired proteins binds to the immobilized ligand, where the other substances are washed through the column by a buffer • The material you want to capture “sticks” to the column and the rest is washed away

23. Types of Chromatography • Gel Filtration Chromatography: also called size exclusion, molecules are separated according to their size and shape

24. Verification • SDS-PAGE Compare protein size to set of sizing standards run

25. SDS Page • Electrophoresis process used for proteins: can determine molecular weight of a protein • The SDS (sodium dodecyl sulfate) helps to unfold protein • Materials Needed for SDS-PAGE • Molecular Weight Markers • 4-20% acrylamide gradient gels • Tris-glycine-SDS buffer • Practice gel loading solution • Marker protein • Sample proteins • Sealant • 50 ml Coomassie Blue Staining Solution (silver stain is most sensitive) • De-staining solution (7.5% acetic acid)and methanol

26. % Acrylamide gels based on MW of protein 7% 50kD to 500kD 10% 20kD to 300kD 12% 10kD to 200kD 15-16% 3kD to 100kD Smaller the protein higher the % gel used Laemmli gels composed of stacking and running gels at different pH Process Unfolds the protein to make it linear Separates the protein and subunits by molecular weight Coats the protein with negative charge (run like gel electrophoresis) use of silver stain for SDS page - Google Videos SDS Page

28. Preserving Proteins • Lyophilization (freeze drying) • First frozen, placed under vacuum to hasten the evaporation of water (I.e. ice crystals go to water vapor). The containers are sealed after the water is removed, leaving the dried proteins behind.

29. Scale-up of Protein Purification • R&D starts with a small-scale level • Production may demand a larger level • Small scale may not be adaptable • If FDA approval has been gained for small-scale, cannot change the parameters when scaled up (so scientists MUST make sure they can scale up before seeking approval)

30. Post-Purification Analysis Methods • Protein Sequencing • X-ray Crystallography

31. 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 chips • Biochips that can be used to identify proteins • Ways to test proteins • Chemical genetics (compare two same species organisms looking for presence and absence of protein) • Gene expression analysis: on/off switch • Protein interaction analysis

32. Protein Engineering • Directed molecular evolution technology