protein purification you are a biochemist working at pharmaceutical company. your boss tells you...

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?

Protein properties

• Molecular weight (size)• pI, isoelectric point (charge)• Solubility (hydrophobicity)• pH, Temp., (stability)• Contaminant properties (proteases)

1

234

5

6

7

8KEEP IT SIMPLE !!

(but be smart !!)

Begin with intact tissue

DisruptBlender, homoginizer

Remove debrisCentrifugation

Precipitate/concentrateAmmonium sulfate

PurifyChromatography

AnalyzeActivity, 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 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”

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

Water

AS

protein

Solution with proteinSolution with protein and AS

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.

Column Chromatography

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

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

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

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

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.

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

Beer’s Law

A = abc = ecl

A

c

x

x

x

xx

*

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

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 GelpH 6.6

Separating GelpH 8.8

Proteins separated by molecular weight

“Ladder”

Myosin – blueb-galactosidase – magentaBovine serum albumin – greenCarbonic anhydrase – violetSoybean trypsin inhibitor – orangeLysozyme – redAprotinin - blue

Kaleidoscope standard

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

chymotrypsin: cleaves at the C-terminal side of aromatic residues, Phe, Tyr, Trp

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.

Quantification of protein, an Enzyme: Activity versus specific activity