Download CHMI 2227E Biochemistry I

CHMI 2227E
Biochemistry I
Protein purification and
characterization
CHMI 2227 - E.R. Gauthier, Ph.D.
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Protein purification
CHMI 2227 - E.R. Gauthier, Ph.D.
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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
CHMI 2227 - E.R. Gauthier, Ph.D.
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Differential precipitation
solubility
1.1. Precipitation by adjusting the pH
pI
CHMI 2227 - E.R. Gauthier, Ph.D.
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http://www.tulane.edu/~biochem/med/pure.htm
Differential precipitation
1.2.Salting out
CHMI 2227 - E.R. Gauthier, Ph.D.
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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%
1)
Pellet: protein A
2)
Add (slowly) (NH4)2SO4 to
20%
Centrifuge to pellet
precipitated proteins
Supernatant: protein B + C
1)
Pellet: protein B
2)
Add (slowly) (NH4)2SO4 to
30%
Centrifuge to pellet
precipitated proteins
Supernatant: protein C
CHMI 2227 - E.R. Gauthier, Ph.D.
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Dialysis
Hours
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Centrifugation
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Protein purification
2. Fine methods
Buffer reservoir
Column
Sample
injector
Pumps
CHMI 2227 - E.R. Gauthier, Ph.D.
Fraction collector
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Protein purification
2.1. Ion exchange chromatography
CHMI 2227 - E.R. Gauthier, Ph.D.
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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
CHMI 2227 - E.R. Gauthier, Ph.D.
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http://www1.amershambiosciences.com/aptrix/upp00919.nsf/Content/LabSep_Prod~SelGuides~Media~GFSelG
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Protein purification
2.2. Molecular sieve chromatography
CHMI 2227 - E.R. Gauthier, Ph.D.
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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
SDS
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Electrophoresis
1. SDS-PAGE Electrophoresis
CHMI 2227 - E.R. Gauthier, Ph.D.
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Electrophoresis
1. SDS-PAGE Electrophoresis
www.biology.ucsd.edu/classes/bggn224.FA06/crash_2.ppt
http://wine1.sb.fsu.edu/bch5425/lect20/lect20.htm
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Electrophoresis
1. SDS-PAGE Electrophoresis
CHMI 2227 - E.R. Gauthier, Ph.D.
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Electrophoresis
Log Mr
1. SDS-PAGE Electrophoresis
Distance migrated from well (cm)
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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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Electrophoresis
4. Western blot analysis
Structure of an antibody
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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
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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
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
Cutting
site
Arg/Lys
C-ter
Phe/Trp/Tyr
C-ter
Asp/Glu
C-ter
Phe/Trp/Tyr
N-ter
Leu/Ile/Trp/Tyr/
Val/Ala/Phe
N-ter
All C-ter a.a.
except Pro,
Arg/Lys
Only Arg/Lys
when C-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
Amino
acid
Cutting site
Cyanogen bromide
Met
C-ter
b-mercaptoethanol
Cys
Disulfide bonds
Iodoacetate
Cys
Prevents 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.
Hydrazine
Destroy all the amino acids
with the exception of the one
at the C-terminus.
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CHMI 2227 - E.R. Gauthier, Ph.D.
Determination of protein sequence
1. Enzyme mapping – example 1
CHMI 2227 - E.R. Gauthier, Ph.D.
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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, Gly2, Lys, Met, Ser,
Thr, Tyr

Chymotrypsin: 2 peptides were
obtained:





Peptide 1: Ala, Gly, Lys, Thr
Peptide 2: Gly, Met, Ser, Tyr
Trypsin: 2 peptides were obtained:




CNBr: 2 peptides were obtained:
Peptide 3: Ala, Gly
Peptide 4: Gly, Lys, Met, Ser, Thr,
Tyr
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?
CHMI 2227 - E.R. Gauthier, Ph.D.
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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
CHMI 2227 - E.R. Gauthier, Ph.D.
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Determination of protein sequence
3. Mass spectrometry
CHMI 2227 - E.R. Gauthier, Ph.D.
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Determination of protein sequence
3. Mass spectrometry
CHMI 2227 - E.R. Gauthier, Ph.D.
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