2011_09_28_protein_purification_crystallization-1

2011_09_28_protein_purification_crystallization-1 - Goals:...

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Goals: • Determine the structure and how it contributes to function • Understand how proteins fold • How do proteins bind their substrates • What is their mechanism of action • What is their function within a cellular context
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Protein purification Step 2: Salting out – some proteins precipitate at high salt concentrations – eg. (NH 4 ) 2 SO 4 - cheap, very soluble, and anti- chaotropic (i.e. stabilizes proteins) - remove excess (NH 4 ) 2 SO 4 using dialysis Rupture cells (sonication) Crude homogenate (protein, cell debris, other stuff) Cells or Tissue Crude Lysate (concentrated protein solution) Step 1: Step 3: Chromatography
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Affinity chromatography • affinity chromatography – separation based on the ability of the protein to carry out specific binding -eg. a plant protein ConA can binds glucose tightly - pass crude extract with ConA over a column of beads with glucose covalently attached to beads - specifically purify ConA from other components that do not bind glucose - ConA eluted by adding increasing amounts of glucose Today: Affinity tags used 1) hexahistidine (His 6 ) - short polypeptide binds to Ni 2+ resin - elute protein with imidazole gradient 2) Glutathione-S-transferase (GST). - small 25 kDa highly soluble protein binds to glutathione resin - elute with reduced glutathione • tags are easily engineered into protein of interest with recombinant techniques • tags are cleaved from protein with engineered enzyme cleavage site such as thrombin
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Gel-filtration chromatography • gel filtration chromatography –separation based on the size (mass) and shape (diameter) of the protein - sample is applied to the top of a column of porous beads about 100 uM in diameter made of a material like dextran or agarose (carbohydrates) eg. Sephadex or sepharose - small molecules can enter the beads, but large ones cannot (small molecules get retarded) - large molecules emerge first, medium size molecules come next, smallest molecules come last - difference in mobility between proteins results in separation - assumptions : a) protein is ~spherical (elongated proteins give larger than expected masses b) no interaction with the sephadex gel - gel filtration chromatography used for 1) purification, 2) desalting (faster than dialysis), 3) to estimate the MW of the protein in its native state (i.e. quaternary structure) Log (MW) Elution Volume x x x x X = standard protein of known MW
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Gel electrophoresis (SDS-PAGE) • SDS-PAGE - test for purity of protein - cheap and little material - SDS coats the protein and denature it - results in all typical proteins having the same negative charge density Protein solution + HSCH 2 CH 2 OH ( ! -ME) + SDS (sodium dodecyl sulphate) heat to denature protein Log (MW) mobility x x x x X = standard protein of known MW - mobility inversely related to MW - gives MW of subunit (chain) - protein travels from negative to positive end Native PAGE: SDS-PAGE without the SDS (non-denaturing) - proteins travel according to charge/mass ratio and conformation
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2011_09_28_protein_purification_crystallization-1 - Goals:...

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