Purification and Protein analysis

Chapter 3 (part 2) Protein purification and Analysis Why purify proteins Pure proteins are required to study enzyme function Pure proteins are required for structural analysis (x-ray crystallography, NMR spectroscopy) Pure proteins are required to obtain amino acid sequence Steps in protein purification Develop assay Choose source of protein Prepare tissue extract cell disruption subcellular fractionation Protein fractionation (several steps) Determination of purity Differential Centrifugation transfer supernatant transfer supernatant transfer supernatant 1000 g 10,000 g 100,000 g tissue homogenate Pellet unbroken cells nuclei chloroplast Pellet mitochondria Pellet microsomal Fraction (ER, golgi, lysosomes, peroxisomes) Super. Cytosol, Soluble enzymes Chromatography Gel Permeation Chromatography Ion-exchange Chromatography + + + + ++ ++ + ++ + ++ + + ++ ++ ++ + + + + + ++ + ++ + + ++ ++ + low salt buffer ++ + + + ++ + + + + + ++ + ++ + + + ++ + + ++ + + ++ ++ + high salt buffer + Cl-+ Cl+ + + + Cl- + + -+ + ++ + Cl + Cl+ + Cl+ + ++ ++ + + + + + ++ + ++ + + ++ ++ - + Affinity Chromatography Add excess ligand SDS poly acrylamide electrophoresis (PAGE) SDS = H3CCH2)10-CH2-OSO3SDS denatures protein coats w/ negative charge - -- - - - - - Used to determine protein MW And purity of protein prep Isoelectric Focusing pH 9 Decreasing pH Decreasing pH - pH 3 + + 2-D Electrophoresis large Decreasing pH Decreasing MW SDS-PAGE Decreasing MW small + Decreasing pH Amino Acid Analysis 1) Acid hydrolyze protein 2) Treat with phenylisothiocyanate (PICT) H N C S S C HN C C O R H O C O- + H 3N C R N 3) Separate derivatized AA's by HPLC Protein Sequencing (Edman Degradation) 1) H O C NH H C R H NH C R O O C X N C S H3N C R Trifluoroacetic acid S R 3) N C HN C C O R H + H 2HN C R O C X Can sequence in 30 to 60 AA's from N-terminus Repeat 2) S N C HN H C O C C X Generate Proteolytic Fragments Endopeptidases Typsin Chymotrypsin Chemical Cleavages Cyanogen Bromide cleaves at COOH end of Met cleaves at COOH end of Lys and Arg cleaves at COOH end of Phe, Tyr, Trp Generate overlapping fragments Sequence individual fragements and piece together sequence Peptide mapping exercise Met-Ala-Arg- Gly-Glu-Tyr-Met-Cys-Lys-Phe-Ala-Glu-Gln-Asp Trypsin Met-Ala-Arg Phe-Ala-Glu-Gln-Asp Gly-Glu-Tyr-Met-Cys-Lys Chymotrysin Met-Ala-Arg- Gly-Glu-Tyr Met-Cys-Lys Phe Ala-Glu-Gln-Asp CNBr Met-Ala-Arg- Gly-Glu-Tyr-Met Cys-Lys -Phe-Ala-Glu-Gln-Asp Proteomic Analysis Continua »