Sinh học - Chapter 3 (part 2): Protein purification and Analysis

Chapter 3 (part 2)Protein purification and AnalysisWhy purify proteins?Pure proteins are required to study enzyme functionPure proteins are required for structural analysis (x-ray crystallography, NMR spectroscopy)Pure proteins are required to obtain amino acid sequence Steps in protein purificationDevelop assay Choose source of proteinPrepare tissue extractcell disruptionsubcellular fractionationProtein fractionation (several steps)Determination of purityDifferential Centrifugationtissuehomogenate1000 gPelletunbroken cellsnucleichloroplasttransfer supernatanttransfer supernatanttransfer supernatant10,000 g100,000 gPelletmitochondriaPelletmicrosomalFraction(ER, golgi,lysosomes,peroxisomes)Super.Cytosol,SolubleenzymesChromatographyGel Permeation Chromatography++++++++++++++++++++++++++++++++++++++++- - -- - -- - -++++++++++++++++++++++++++++++++++++++++++++++++++++++++++- - -- - -- - -Cl-Cl-Cl-Cl-Cl-Cl-++++++++++++++++++++++++++++++++++++++++- - -- - -- - -++++++++++++++++++low salt bufferhigh salt bufferIon-exchange ChromatographyAffinity ChromatographyAdd excess ligandSDS poly acrylamide electrophoresis (PAGE)SDS = H3C-(CH2)10-CH2-OSO3-----------------SDS – denatures proteincoats w/ negative chargeUsed to determine protein MWAnd purity of protein prepIsoelectric FocusingDecreasing pH+-Decreasing pH+-pH 9pH 32-D ElectrophoresisDecreasing MWsmalllargeDecreasing pH+-SDS-PAGEDecreasing pHDecreasing MWAmino Acid AnalysisAcid hydrolyze proteinTreat with phenylisothiocyanate (PICT)Separate derivatized AA’s by HPLC+Protein Sequencing (Edman Degradation)+Trifluoroacetic acid1)2)3)RepeatCan sequence in 30 to 60 AA’s from N-terminusGenerate Proteolytic FragmentsEndopeptidasesTypsin cleaves at COOH end of Lys and ArgChymotrypsin cleaves at COOH end of Phe, Tyr, TrpChemical Cleavages Cyanogen Bromide cleaves at COOH end of MetGenerate overlapping fragmentsSequence individual fragments and piece together sequence Peptide mapping exerciseMet-Ala-Arg- Gly-Glu-Tyr-Met-Cys-Lys-Phe-Ala-Glu-Gln-AspTrypsinMet-Ala-ArgPhe-Ala-Glu-Gln-AspGly-Glu-Tyr-Met-Cys-LysChymotrysinMet-Ala-Arg- Gly-Glu-TyrMet-Cys-Lys –PheAla-Glu-Gln-AspCNBrMetAla-Arg-Gly-Glu-Tyr-MetCys-Lys-Phe-Ala-Glu-Gln-AspProteomic AnalysisMatrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF)