Protein Purification
You are a biochemist working at pharmaceutical
company. Your boss tells you that we are starting to
research metabolism in cows. As it turns out, a
hormone enhancing drug that was given to the cows is
suspected to increase the metabolism! The company
thinks that the drug might be activating the enzyme
Lactate Dehydrogenase (LDH).
You, the biochemist, needs to isolate this 1 enzyme to
study it....
Is this possible?
Protein properties
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•
•
•
•
Molecular weight (size)
pI, isoelectric point (charge)
Solubility (hydrophobicity)
pH, Temp., (stability)
Contaminant properties (proteases)
1
2
3
4
5
6
7
8
KEEP IT SIMPLE !!
(but be smart !!)
Begin with intact tissue
Disrupt
Blender, homoginizer
Remove debris
Centrifugation
Precipitate/concentrate
Ammonium sulfate
Purify
Chromatography
Analyze
Activity, molecular weight
Protein Purification!
AS precipitation
(NH4)2SO4
Very cheap! Very soluble in H2O
Relies on fact that proteins loose solubility as concentration of salt
is increased
When protein precipitates is characteristic of particular protein
Results in a partial purification of all proteins with similar
solubility characteristics
Can rid solution of other non-proteins also!
Produces “salt cuts”
Salting out
At high concentrations added salt lowers the solubility
of macromolecules because it competes for the solvent
(H2O) needed to solvate the macromolecules.
Salt interacts with water via electrostatic interactions
So high [salt] removes the solvation sphere from the
protein molecules and they come out of solution.
Proteins interact with other proteins via hydrophobic
interactions
Solution with protein
Solution with protein and AS
Water
AS
protein
Dialysis
Passage of solutes through a semi-permeable membrane.
Pores in the dialysis membrane are of a certain size.
Protein stays in; water, salts, protein fragments, and other
molecules smaller than the pore size pass through.
Column Chromatography
Columns
Common types of column chromatography:
Ion-exchange chromatography - separation
based upon the overall charge of molecules
Gel-filtration chromatography - separation
based upon molecular size
Affinity chromatography - separation by
specific binding interactions between column
matrix and target proteins
Ion-exchange chromatography
KCl + H2O  K+ + Cl-
cation exchange chromatography: positively charged ions bind to a
negatively charged resin
anion exchange chromatography: negatively charged bind to a
positively charged resin
Anion Exchange Chromatography
Cation Exchange Chromatography
Gel filtration chromatography
Affinity Chromatography
Affinity Chromatography
resin have Ni++
attached
Solvent flow
His tag on protein
binds to Ni++
his
Elute with imidazole
histidine
Ni++
imidazole
Ni++
Ni++
Ni++
Ni++
HPLC
High Performance Liquid Chromatography: can be
applied to many different resins
Most common: separation is based on the molecule’s
relative solubility in H2O or polarity
Material will be eluted with a gradient of non-polar
solvent
Protein Concentration
Lowry ( most cited reference in biology)
Color assay
A280
Intrinsic absorbance
Relies on aromatic amino acids
Bradford
Shifts Amax of dye from 465nm to 595nm
Lowry, OH, NJ Rosbrough, AL Farr, and RJ Randall. J. Biol. Chem. 193: 265.
1951.
Beer’s Law
Beer’s Law is stated in a way to make certain
quantities easy to compare and interpret.
Parameters:
l – sample pathlength (usually 1cm)
c – concentration (M)
e – molar absorption coefficient
A – light intensity (absorbance)
A=ecl
Beer’s Law
A = abc = ecl
x
x
A
x
*
x
x
c
What:
sample
dilute by ½
dilute by ½ again
dilute by ½
Conc:
1g/mL
1/2g/mL
1/4g/mL
1/8g/mL
Abs:
2.0
1.0
0.5
0.25
Beer's Law
2.5
Absorbance
2
1.5
Series1
1
0.5
0
0
0.2
0.4
0.6
Concentration
0.8
1
1.2
Absorbance
Concentration
2
1
1
0.5
0.5
0.25
0.25
0.125
Where’s the protein?
A280
Uses intrinsic absorbance
Detects Y and W residues and little S-S
Depends on protein structure, native state and AA composition
Retains protein function
Layne, E. Spectrophotometric and Turbidimetric Methods for Measuring Proteins.
Methods in Enzymology 3: 447-455. 1957.
What is SDS-PAGE?
Polyacrylamide gel electrophoresis (PAGE)
Separates molecules on a polyacrylamide
gel matrix when an electric field is applied
SDS-PAGE. Sodium dodecyl sulfate (SDS)
coats proteins with negative charges. Coated
polypeptide chains then separate by
molecular mass (method to determine
molecular weight)
Why do we need to denature
the proteins?
gels
Dr Caran JMU Chemistry
(a) SDS-PAGE Electrophoresis
(b) Protein banding pattern after run
pH 8.3
Stacking Gel
pH 6.6
Separating Gel
pH 8.8
Proteins separated by molecular weight
“Ladder”
Kaleidoscope standard
Myosin – blue
b-galactosidase – magenta
Bovine serum albumin – green
Carbonic anhydrase – violet
Soybean trypsin inhibitor – orange
Lysozyme – red
Aprotinin - blue
Proteases will cleave amide bonds at specific locations
Then the puzzle can be solved!
Proteases (peptidases): Enzymes that catalyzed the hydrolysis
of the amide bonds of peptides and proteins.
trypsin: cleaves at the C-terminal side of Arg, Lys
chymotrypsin: cleaves at the C-terminal side of aromatic residues,
Phe, Tyr, Trp
Align the sequences of the peptide fragments from the two
complementary cleavage methods.
E-A-Y-L-V-C-G-E-R
F-V-N-Q-H-L-F-S-H-L-K
G-C-F-L-P-K
L-G-A
F-V-N-Q-H-L-F
S-H-L-K-E-A-Y
L-V-C-G-E-R-G-C-F
L-P-K-L-G-A
F-V-N-Q-H-L-F
F-V-N-Q-H-L-F-S-H-L-K
S-H-L-K-E-A-Y
E-A-Y-L-V-C-G-E-R
L-V-C-G-E-R-G-C-F
G-C-F-L-P-K
L-P-K-L-G-A
F-V-N-Q-H-L-F-S-H-L-K-E-A-Y-L-V-C-G-E-R-G-C-F-L-P-K-L-G-A
Quantification of protein, an Enzyme:
Activity versus specific activity