Card Set Information

2010-12-02 16:46:34

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  1. Proteins
    • polymers of Amino Acids
    • Specific shapes
    • specific functions
    • structure- function relationships
  2. Proteins as BT products
    • Enzymes- biological catalysts - speeds up chemical reactions
    • Hormones - chemical messengers (sends signals)
    • Antibodies - proteins that bind and neutralize foreign molecules (immune system)
  3. Enzymes
    • Involved in many production processes
    • -Chymosin: an enzyme involved in producing casein (milk/cheese protein) -- food processing
    • textlies/leather goods; Creation of the wool/leather, stone-washed jeans
    • detergents; proteases lipases amylases
  4. Hormones
    Erythropoietins; stimulate blood production (people with anemia; kidney disease)

    Insulin; used to treat Type 1 diabetes ( for peple who can't regulate glucose pancreas)
  5. Monclonial antibodies
    • Used to treat disease
    • a number are now on the market
    • Humira TM; Adelimumab, used to treat rheumatoid arthritis and other conditions
    • -TNF inhibitor ( an anti-inflammatory agent)
    • -A "blockbuster" drug ( >$1 billion in sales/year)
    • antibodies attack foreign tissue; protein
  6. Protein structure
    • structure determines function
    • four levels of protein structure
    • If protein doesn't fold properly it wont work properly
  7. Polypeptide
    • a long polymer of amino acids
    • a protein is a functional polypeptide that might have other things associated with it
  8. Primary Structure
    • The linear amino acid sequence of the polypeptide
    • 20 common amino acids
    • a change in 1 amino acid in the sequence might make the protein lose all function
  9. Secondary structure
    • the primary structure folds , just barely, into alpha helices or beta pleated sheets
  10. Tertiary structure
    • the polypeptide folds into a specfic 3D structure
    • certain attractions are present between the alpha helices and pleated sheets causing the bending and twisting into shape
  11. Quarternary structure
    • More than 1 polypeptide chain associates in 3D structure
    • lustering
    • of several individual peptide or protein chains into a final
    • specific shape. A variety of bonding interactions including hydrogen
    • bonding, salt bridges, and disulfide bonds hold the various chains
    • into a particular geometry. There are two major categories of proteins with quaternary structure - fibrous and globular.
  12. Protein Production
    • Two phases
    • Upstream processing (getting proteins to express) {proteins are produced by cells genetically engineered to contain the human gene
    • which will express the protein of interest}
    • Downstream processing (recovery and purification of biosynthetic products)
  13. What expresses your protein?
    • Microorganisms
    • -Bacteria
    • -fungi (yeast)
    • Advantages
    • -cheap and easy to grow in large fermentors
    • Problems
    • - Bacteria don't modify mammalian proteins "correctly" (don't fold properly)
    • bacteria don't put sugar on cells, if sugar is important use mammalian cells
    • Neither do yeast, but they are a little better
  14. Expression in cell culture
    • Insect cells - sugars are still not the same as mammalian cells.
    • Mammalian cells ex; CHO cells , Human embryonic kidney (HEK)
    • Advantages - proper processing of proteins
    • Disadvantages - Expensive to grow -Difficult to scale up (bioreactors)
  15. Expression in whole animals
    • Farming
    • Transgenic goats or cows - insulin in cows; milk cows for insulin using a milk promoter
    • Transgenic plant -tobacco
  16. Making the extract ( Downstreaming)
    • For secreted protein, media is collected or
    • Whole cells are broken open
  17. Prevent degradation, denaturation (Protein loses its shape like an egg when cooked)
    • Keep protein extract cold
    • remove or inhibit proteases ( chop iup protein)
    • Don't create foam, minimize shear forces
    • (gentle handling ) dont shake
  18. Separate the proteins
    • protein precipitation (add salt for concentration to increase, protein falls out of solution to the bottom of tube, becomes white , you must know the concentration your protein alone will respond to. After removing soln add media to precipitation to dissolve.
    • Selective precipitation can remove proteins that arent the one you want, or preciptate yours (and then redissolve and renature)
  19. Filtration
    • Size-based separation (holea so small it can separate proteins from each other)
    • centrifugation
    • Membrane filtration
    • microfiltration
    • Ultrafiltration
  20. Chromatography
    • Several methods commonly used
    • Often done in a column; then called column chromatography
  21. Size exclusion chromatography
    • SEC
    • Gel filtration chromatography
    • *Tiny beads with tiny holes
    • separates proteins by size
    • Smallest proteins travel fastest through the column
    • hplc= high pressure chromotography
  22. Ion exchange chromatography
    • IEC
    • Separates proteins by charge
    • Different types of IEC resins separate proteins differently (but using same principles)
    • * column with a positive charge the negative ones will stick to it and the positive charge will go through
  23. Affinity Chromatography
    • Very specific- relies on ability of a protein to specifically bind a substance (or antibody)
    • Terminology
    • Ligand : a molecule that specifically binds something els ( protein)
    • Ex: use an antibody against your protein
    • Pass a mixture over the AB column -Only your protein sticks; everything else passes through
    • Wash column
    • elute your protein
  24. Electrophoresis
    • Another technique that can separate a mixture of proteins
    • Native gel electrophoresis -separates proteins based on charge
    • SDS-PAGE (SDS poluacrylamide gel electrophoresis) -separates proteins based on size
    • Isoelectric focusing(IEF) - separates proteins based on isoelectric point
  25. How to purify a protein
    Combine methods in a process that separates your protein from the rest of them without inactivating your protein
  26. Analytical Methods
    Used to check purity of proein (among other uses)
  27. HPLC (High pressure Liquid Chromatography)
    • High performance liquid chromatography
    • Chromatography (already covered) UNDER PRESSURE (faster)
    • FPLC (fast performance LC) * not as good as HPLC but faster
  28. Mass Spectrometry
    • Mass Spec or MS(measures the mass of the protein)
    • Very sensitive method to determine the mass of a small protein
    • Lots of "alphabet soup" here
    • - MALDI-TOF
    • LC/MS, EC-MS, MS/MS
  29. 2d Gell electrophoresis
    (separates proein in 2 Dimensions by charge
    • combines 2 electrophoresis techniques
    • IEF and SDS-PAGE
    • (Sodium D SULFATE) strongly negative molecule
    • that sticks across protein and straightens it out to be able to measure its size
    • Proteins with more positive charge will migrate to bottom and Vice Versa
  30. 2D gel of pure protein
  31. Stabilize/Preserve protein
    • Lyophilization -freeze-drying
    • Store in solution - will likely have to be kept at 4 deg C
    • Freezing - Not the best way to package your protein
    • A rule of thumb is to limit freeze-thaw cycles to 3