CHMI E.R. Gauthier, Ph.D. 1 CHMI 2227E Biochemistry I Protein purification and characterization

CHMI E.R. Gauthier, Ph.D.2 Protein purification

CHMI E.R. Gauthier, Ph.D.3 Protein purification General procedure Homogenization Crude protein extract Coarse purification steps Chromatography 1, 2, 3….x steps PURE! (well, let’s hope so…) Monitor presence of protein of interest: - Activity - Purity - Quantity

CHMI E.R. Gauthier, Ph.D.4 Protein purification 1. Coarse methods

CHMI E.R. Gauthier, Ph.D.5 Differential precipitation 1.1. Precipitation by adjusting the pH pH solubility pI

CHMI E.R. Gauthier, Ph.D.6 Differential precipitation 1.2.Salting out

CHMI E.R. Gauthier, Ph.D.7 Differential precipitation 1.2.Salting out Mixture of proteins in buffer: A: precipitates at salt [ ] = 15% B: precipitates at salt [ ] = 25 % C: precipitates at salt [ ] = 35% 1)Add (slowly) (NH 4 ) 2 SO 4 to 20% 2)Centrifuge to pellet precipitated proteins Pellet: protein A Supernatant: protein B + C 1)Add (slowly) (NH 4 ) 2 SO 4 to 30% 2)Centrifuge to pellet precipitated proteins Pellet: protein B Supernatant: protein C

CHMI E.R. Gauthier, Ph.D.8 Dialysis Hours

CHMI E.R. Gauthier, Ph.D.9 Centrifugation

CHMI E.R. Gauthier, Ph.D.10 Protein purification 2. Fine methods Column Fraction collector Buffer reservoir Pumps Sample injector

CHMI E.R. Gauthier, Ph.D.11 Protein purification 2.1. Ion exchange chromatography

CHMI E.R. Gauthier, Ph.D.12 Protein purification 2.1. Ion exchange chromatography DesorptionAdsorption Anion exchanger NaCl Example of cation exchange chromatography

CHMI E.R. Gauthier, Ph.D.13 Protein purification 2.1. Ion exchange chromatography Progressive, linear change in NaCl concentration Stepwise gradient of NaCl concentration Add buffer

CHMI E.R. Gauthier, Ph.D.14 Protein purification 2.2. Molecular sieve chromatography

CHMI E.R. Gauthier, Ph.D.15

CHMI E.R. Gauthier, Ph.D.16 Protein purification 2.2. Molecular sieve chromatography

CHMI E.R. Gauthier, Ph.D.17 Protein purification 2.3. Affinity chromatography

CHMI E.R. Gauthier, Ph.D.18 Analysis of proteins Electrophoresis

CHMI E.R. Gauthier, Ph.D.19 Electrophoresis 1. SDS-PAGE Electrophoresis SDS

CHMI E.R. Gauthier, Ph.D.20 Electrophoresis 1. SDS-PAGE Electrophoresis

CHMI E.R. Gauthier, Ph.D.21 Electrophoresis 1. SDS-PAGE Electrophoresis

CHMI E.R. Gauthier, Ph.D.22 Electrophoresis 1. SDS-PAGE Electrophoresis

CHMI E.R. Gauthier, Ph.D.23 Electrophoresis 1. SDS-PAGE Electrophoresis Log Mr Distance migrated from well (cm)

CHMI E.R. Gauthier, Ph.D.24 Electrophoresis 2. Isoelectrofocusing

CHMI E.R. Gauthier, Ph.D.25 Electrophoresis 2. Isoelectrofocusing pH in gel = pI of proteins

CHMI E.R. Gauthier, Ph.D.26 Electrophoresis 3. Two-dimensional gel electrophoresis

CHMI E.R. Gauthier, Ph.D.27 Electrophoresis 3. Two-dimensional gel electrophoresis Each spot is a single protein

CHMI E.R. Gauthier, Ph.D.28 Electrophoresis 4. Western blot analysis Structure of an antibody

CHMI E.R. Gauthier, Ph.D.29 Electrophoresis 4. Western blot analysis

CHMI E.R. Gauthier, Ph.D.30 Protein purification Example and data analysis

CHMI E.R. Gauthier, Ph.D.31 Protein purification Example and data analysis Coomassie-blue-stained PAGE-SDS gel

CHMI E.R. Gauthier, Ph.D.32 Protein purification Example and data analysis

CHMI E.R. Gauthier, Ph.D.33 Protein sequencing

CHMI E.R. Gauthier, Ph.D.34 Protein sequence - Sickle-cell anemia Normal red blood cell Sickled red blood cell

CHMI E.R. Gauthier, Ph.D.35 Determination of protein sequence 1. Enzyme mapping EnzymeAmino acidCutting site TrypsinArg/LysC-ter ChymotrypsinPhe/Trp/TyrC-ter Protease V8Asp/GluC-ter PepsinPhe/Trp/TyrN-ter ThermolysinLeu/Ile/Trp/Tyr/ Val/Ala/Phe N-ter Carboxypeptidase A All C-ter a.a. except Pro, Arg/Lys - Free amino acids from the C-ter - Doesn’t cut if Pro is the penultimate amino acid Carboxypeptidase B Only Arg/Lys when C-ter Based on the property of some enzymes to cut the peptide bonds next to specific amino acids; ChemicalAmino acid Cutting site Cyanogen bromideMetC-ter  -mercaptoethanol CysDisulfide bonds IodoacetateCysPrevents the reduction of disulfide bonds 1) 1-Fluoro-2,4 dinitrobenzene (FDNB) 2) Dansyl chloride 3) Dabsyl chloride Destroy all the amino acids with the exception of the one at the N-terminus. HydrazineDestroy all the amino acids with the exception of the one at the C-terminus. NOTE: Trypsin, Chymotrypsin, protease V8, pepsin and thermolysin do NOT cut if Pro is part of the peptide bond.

CHMI E.R. Gauthier, Ph.D.36 Determination of protein sequence 1. Enzyme mapping – example 1

CHMI E.R. Gauthier, Ph.D.37 Determination of protein sequence 1. Enzyme mapping – example 2  HCl 6M: Ala, Gly 2, Lys, Met, Ser, Thr, Tyr  CNBr: 2 peptides were obtained: Peptide 1: Ala, Gly, Lys, Thr Peptide 2: Gly, Met, Ser, Tyr  Trypsin: 2 peptides were obtained: Peptide 3: Ala, Gly Peptide 4: Gly, Lys, Met, Ser, Thr, Tyr  Chymotrypsin: 2 peptides were obtained: Peptide 5: Gly, Tyr Peptide 6: Ala, Gly, Lys, Met, Ser, Thr  FDNB: yields Gly  Carboxypeptidase A: yields Gly The following data were obtained after treating an octopeptide with the following reagents: What is the sequence of this peptide?

CHMI E.R. Gauthier, Ph.D.38 Determination of protein sequence 2. Edman degradation

CHMI E.R. Gauthier, Ph.D.39 Determination of protein sequence 2. Edman degradation Identify

CHMI E.R. Gauthier, Ph.D.40 Determination of protein sequence 3. Mass spectrometry

CHMI E.R. Gauthier, Ph.D.41 Determination of protein sequence 3. Mass spectrometry

CHMI E.R. Gauthier, Ph.D.42 Determination of protein sequence 3. Mass spectrometry