Campbell6e lecture ch5


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Campbell6e lecture ch5

  1. 1. Chapter Five Protein Purification and Characterization Techniques
  2. 2. Isolation of Proteins from Cells <ul><li>Many different proteins exists within one cell </li></ul><ul><ul><li>• Many steps needed to extract protein of interest, and separate from many contaminants </li></ul></ul><ul><ul><li>• Before purification begins, protein must be released from cell by homogenization </li></ul></ul>
  3. 3. How We Get Proteins Out of Cells
  4. 4. Salting Out <ul><li>• After Proteins solubilized, they can be purified based on solubility (usually dependent on overall charge, ionic strength, polarity) </li></ul><ul><li>• Ammonium sulfate (NH 4 SO 4 ) commonly used to “salt out” </li></ul><ul><li>• Takes away water by interacting with it, makes protein less soluble because hydrophobic interactions among proteins increases </li></ul><ul><li>• Different aliquots taken as function of salt concentration to get closer to desired protein sample of interest ( 30, 40, 50, 75% increments ) </li></ul><ul><li>• One fraction has protein of interest </li></ul>
  5. 5. Differential Centrifugation <ul><li>• Sample is spun, after lysis, to separate unbroken cells, nuclei, other organelles and particles not soluble in buffer used </li></ul><ul><li>• Different speeds of spin allow for particle separation </li></ul>
  6. 6. Column Chromatography <ul><li>• Basis of Chromatography </li></ul><ul><ul><li>Different compounds distribute themselves to a varying extent between different phases </li></ul></ul><ul><li>• Interact/distribute themselves </li></ul><ul><li>• In different phases </li></ul><ul><li>• 2 phases: </li></ul><ul><li>• Stationary : samples interacts with this phase </li></ul><ul><li>• Mobile : Flows over the stationary phase and carries along with it the sample to be separated </li></ul>
  7. 7. Column Chromatography
  8. 8. Size-Exclusion/Gel-Filtration <ul><li>• Separates molecules based on size. </li></ul><ul><li>• Stationary phase composed of cross-linked gel particles. </li></ul><ul><li>• Extent of cross-linking can be controlled to determine pore size </li></ul><ul><li>• Smaller molecules enter the pores and are delayed in elution time. Larger molecules do not enter and elute from column before smaller ones. </li></ul>
  9. 9. Size Exclusion/Gel-filtration (Cont’d)
  10. 10. Affinity Chromatography <ul><li>• Uses specific binding properties of molecules/proteins </li></ul><ul><li>• Stationary phase has a polymer that can be covalently linked to a compound called a ligand that specifically binds to protein </li></ul>
  11. 11. Ion Exchange <ul><li>• Interaction based on overall charge (less specific than affinity) </li></ul><ul><li>• Cation exchange </li></ul><ul><li>• Anion exchange </li></ul>
  12. 12. Electrophoresis <ul><li>• Electrophoresis - charged particles migrate in electric field toward opposite charge </li></ul><ul><li>• Proteins have different mobility: </li></ul><ul><ul><li>Charge </li></ul></ul><ul><ul><li>Size </li></ul></ul><ul><ul><li>Shape </li></ul></ul><ul><li>• Agarose used as matrix for nucleic acids </li></ul><ul><li>• Polyacrylamide used mostly for proteins </li></ul>
  13. 13. Electrophoresis (Cont’d) <ul><li>• Polyacrylamide has more resistance towards larger molecules than smaller </li></ul><ul><li>• Protein is treated with detergent (SDS) sodium dodecyl sulfate </li></ul><ul><li>• Smaller proteins move through faster (charge and shape usually similar) </li></ul>
  14. 14. Isoelectric Focusing <ul><li>• Isolectric focusing- based on differing isoelectric pts. (pI) of proteins </li></ul><ul><li>• Gel is prepared with pH gradient that parallels electric-field. What does this do? </li></ul><ul><li>• Charge on the protein changes as it migrates. </li></ul><ul><li>• When it gets to pI, has no charge and stops </li></ul>
  15. 15. Primary Structure Determination <ul><li>How is 1˚ structure determined? </li></ul><ul><li>Determine which amino acids are present (amino acid analysis) </li></ul><ul><li>Determine the N- and C- termini of the sequence (a.a sequencing), and the Internal Residues </li></ul><ul><li>Determine the sequence of smaller peptide fragments (most proteins > 100 a.a) </li></ul><ul><li>4) Some type of cleavage into smaller units necessary </li></ul>
  16. 16. Primary Structure Determination
  17. 17. Protein Cleavage <ul><li>Protein cleaved at specific sites by: </li></ul><ul><li>Enzymes- Trypsin, Chymotrypsin, Carboxypeptidases (C-terminus) </li></ul><ul><li>Chemical reagents </li></ul><ul><li>Cyanogen bromide, cleaves at Methionine; </li></ul><ul><li>PITC, cleaves from N-terminus (Edman Degradation) </li></ul><ul><li>Hydrazine, cleaves from C-terminus </li></ul><ul><li>Enzymes which cleaves Internal Residues: </li></ul><ul><li>Trypsin- Cleaves @ C-terminal of (+) charged side chains (basic amino acid) </li></ul><ul><li>Chymotrypsin- Cleaves @ C-terminal of aromat ics </li></ul>
  18. 18. Peptide Digestion
  19. 19. Cleavage by CnBr <ul><li>Cleaves @ C-terminal of INTERNAL methionines </li></ul>
  20. 20. Determining Protein Sequence <ul><li>After cleavage, mixture of peptide fragments produced. </li></ul><ul><li>• Can be separated by HPLC or other chromatographic techniques </li></ul><ul><li>• Use different cleavage reagents to help in 1˚ determination </li></ul>
  21. 21. Peptide Sequencing <ul><li>• Can be accomplished by Edman Degradation </li></ul><ul><li>• Relatively short sequences (30-40 amino acids) can be determined quickly </li></ul><ul><li>• So efficient, today N-/C-terminal residues usually not done by enzymatic/chemical cleavage </li></ul>
  22. 22. Peptide Sequencing