Liver MSc Clinical Biochemistry Dr Sarah Mapplebeck Consultant Clinical Biochemist.
Protein Purification You are a biochemist working at pharmaceutical company. Your boss tells you...
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Transcript of Protein Purification You are a biochemist working at pharmaceutical company. Your boss tells you...
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Protein PurificationYou 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?
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Protein properties
• Molecular weight (size)• pI, isoelectric point (charge)• Solubility (hydrophobicity)• pH, Temp., (stability)• Contaminant properties (proteases)
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1
234
5
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8KEEP IT SIMPLE !!
(but be smart !!)
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Begin with intact tissue
DisruptBlender, homoginizer
Remove debrisCentrifugation
Precipitate/concentrateAmmonium sulfate
PurifyChromatography
AnalyzeActivity, molecular weight
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Protein Purification!
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AS precipitation(NH4)2SO4
Very cheap! Very soluble in H2O
Relies on fact that proteins loose solubility as concentration of salt is increasedWhen protein precipitates is characteristic of particular proteinResults in a partial purification of all proteins with similar
solubility characteristicsCan rid solution of other non-proteins also!
Produces “salt cuts”
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Salting outAt 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
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Water
AS
protein
Solution with proteinSolution with protein and AS
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DialysisPassage 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.
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Column Chromatography
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ColumnsCommon 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
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Ion-exchange chromatography
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KCl + H2O K+ + Cl-
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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
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Gel filtration chromatography
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Affinity Chromatography
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resin have Ni++ attached
His tag on protein binds to Ni++
Elute with imidazole
Ni++
Ni++
Ni++
his
Ni++
Ni++
Solvent flow
histidine imidazole
Affinity Chromatography
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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
HPLC
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Protein ConcentrationLowry ( most cited reference in biology)
Color assayA280
Intrinsic absorbanceRelies on aromatic amino acids
BradfordShifts Amax of dye from 465nm to 595nm
Lowry, OH, NJ Rosbrough, AL Farr, and RJ Randall. J. Biol. Chem. 193: 265. 1951.
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Beer’s LawBeer’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
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Beer’s Law
A = abc = ecl
A
c
x
x
x
xx
*
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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
Absorbance Concentration2 11 0.5
0.5 0.250.25 0.125
Beer's Law
0
0.5
1
1.5
2
2.5
0 0.2 0.4 0.6 0.8 1 1.2
Concentration
Abso
rban
ce
Series1
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Where’s the protein?
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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.
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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)
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Why do we need to denature the proteins?
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gels
Dr Caran JMU Chemistry
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(a) SDS-PAGE Electrophoresis (b) Protein banding pattern after run
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pH 8.3
Stacking GelpH 6.6
Separating GelpH 8.8
Proteins separated by molecular weight
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“Ladder”
Myosin – blueb-galactosidase – magentaBovine serum albumin – greenCarbonic anhydrase – violetSoybean trypsin inhibitor – orangeLysozyme – redAprotinin - blue
Kaleidoscope standard
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Proteases will cleave amide bonds at specific locations Then the puzzle can be solved!
Proteases (peptidases): Enzymes that catalyzed the hydrolysisof the amide bonds of peptides and proteins.
trypsin: cleaves at the C-terminal side of Arg, Lys
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chymotrypsin: cleaves at the C-terminal side of aromatic residues, Phe, Tyr, Trp
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E-A-Y-L-V-C-G-E-RF-V-N-Q-H-L-F-S-H-L-KG-C-F-L-P-KL-G-A
F-V-N-Q-H-L-FS-H-L-K-E-A-YL-V-C-G-E-R-G-C-FL-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
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
Align the sequences of the peptide fragments from the two complementary cleavage methods.
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Quantification of protein, an Enzyme: Activity versus specific activity
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