Transcript purification
CHMI 2227E Biochemistry I
Protein purification and characterization CHMI 2227 - E.R. Gauthier, Ph.D.
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Protein purification
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Protein purification
General procedure
Crude protein extract Homogenization Monitor presence of protein of interest: - Activity - Purity - Quantity PURE!
(well, let’s hope so…) CHMI 2227 - E.R. Gauthier, Ph.D.
Coarse purification steps Chromatography 1, 2, 3….x steps
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Protein purification
1. Coarse methods
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Differential precipitation
1.1. Precipitation by adjusting the pH
CHMI 2227 - E.R. Gauthier, Ph.D.
pI pH
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Differential precipitation
1.2.Salting out
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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% Pellet: protein A 1) 2) Add (slowly) (NH 4 ) 2 SO 4 20% to Centrifuge to pellet precipitated proteins Supernatant: protein B + C Pellet: protein B 1) 2) Add (slowly) (NH 4 ) 2 SO 4 30% to Centrifuge to pellet precipitated proteins Supernatant: protein C CHMI 2227 - E.R. Gauthier, Ph.D.
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Dialysis
Hours CHMI 2227 - E.R. Gauthier, Ph.D.
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Centrifugation
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Protein purification
2. Fine methods
Buffer reservoir Sample injector Column Pumps CHMI 2227 - E.R. Gauthier, Ph.D.
Fraction collector
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Protein purification
2.1. Ion exchange chromatography
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Protein purification
2.1. Ion exchange chromatography
Adsorption Desorption NaCl Anion exchanger Example of cation exchange chromatography CHMI 2227 - E.R. Gauthier, Ph.D.
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Protein purification
2.1. Ion exchange chromatography
Progressive,
linear
change in NaCl concentration Add buffer Stepwise gradient of NaCl concentration CHMI 2227 - E.R. Gauthier, Ph.D.
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Protein purification
2.2. Molecular sieve chromatography
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Protein purification
2.2. Molecular sieve chromatography
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Protein purification
2.3. Affinity chromatography
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Analysis of proteins
Electrophoresis
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Electrophoresis
1. SDS-PAGE Electrophoresis
CHMI 2227 - E.R. Gauthier, Ph.D.
SDS 19
Electrophoresis
1. SDS-PAGE Electrophoresis
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Electrophoresis
1. SDS-PAGE Electrophoresis
http://wine1.sb.fsu.edu/bch5425/lect20/lect20.htm
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Electrophoresis
1. SDS-PAGE Electrophoresis
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Electrophoresis
1. SDS-PAGE Electrophoresis
Distance migrated from well (cm) CHMI 2227 - E.R. Gauthier, Ph.D.
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Electrophoresis
2. Isoelectrofocusing
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Electrophoresis
2. Isoelectrofocusing
pH in gel = pI of proteins CHMI 2227 - E.R. Gauthier, Ph.D.
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Electrophoresis
3. Two-dimensional gel electrophoresis
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Electrophoresis
3. Two-dimensional gel electrophoresis
Each spot is a single protein
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CHMI 2227 - E.R. Gauthier, Ph.D.
Electrophoresis
4. Western blot analysis
Structure of an antibody CHMI 2227 - E.R. Gauthier, Ph.D.
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Electrophoresis
4. Western blot analysis
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Protein purification
Example and data analysis
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Protein purification
Example and data analysis
Coomassie-blue-stained PAGE-SDS gel CHMI 2227 - E.R. Gauthier, Ph.D.
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Protein purification
Example and data analysis
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Protein sequencing
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Protein sequence - Sickle-cell anemia Normal red blood cell CHMI 2227 - E.R. Gauthier, Ph.D.
Sickled red blood cell
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Determination of protein sequence
1. Enzyme mapping
Enzyme
Trypsin Chymotrypsin Protease V8 Pepsin Thermolysin Carboxypeptidase A Carboxypeptidase B
Amino acid
Arg/Lys Phe/Trp/Tyr Asp/Glu Phe/Trp/Tyr Leu/Ile/Trp/Tyr/ Val/Ala/Phe All C-ter a.a. except Pro, Arg/Lys Only Arg/Lys when C-ter
Cutting site
C-ter C-ter C-ter N-ter N-ter - Free amino acids from the C-ter Doesn’t cut if
Pro
is the penultimate amino acid
NOTE
: Trypsin, Chymotrypsin, protease V8, pepsin and thermolysin do NOT cut if
Pro
is part of the peptide bond.
Based on the property of some enzymes to cut the peptide bonds next to specific amino acids;
Chemical
Cyanogen bromide b -mercaptoethanol Iodoacetate 1) 1-Fluoro-2,4 dinitrobenzene (FDNB) 2) Dansyl chloride 3) Dabsyl chloride Hydrazine CHMI 2227 - E.R. Gauthier, Ph.D.
Amino acid
Met
Cutting site
C-ter Cys Disulfide bonds Cys Prevents the reduction of disulfide bonds Destroy all the amino acids with the exception of the one at the
N-terminus
.
Destroy all the amino acids with the exception of the one at the
C-terminus
.
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Determination of protein sequence
1. Enzyme mapping – example 1
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Determination of protein sequence
1. Enzyme mapping – example 2
The following data were obtained after treating an octopeptide with the following reagents: 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 What is the sequence of this peptide?
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Determination of protein sequence
2. Edman degradation
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Determination of protein sequence
2. Edman degradation
Identify CHMI 2227 - E.R. Gauthier, Ph.D.
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Determination of protein sequence
3. Mass spectrometry
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Determination of protein sequence
3. Mass spectrometry
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Determination of protein sequence
3. Mass spectrometry
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