Protein Purification

Levels of Structure in Protein

• Primary: A description of all covalent bonds. The sequence of AA residues

• Secondary: particularly stable arrangements of AA giving rise to recurring structural patterns.

• Tertiary: All aspects of the 3D folding of a polypeptide.• Quaternary: The spatial arrangement of multisubunits

protein

Protein Purification

• Crude extract: breaking cells, by osmosis lysis or homogenization.

• Fractionation: separate proteins into different fraction based on size of charge.

• Salting out: The solubility of proteins is lowered at high salt concentration. Ammonium sulfate ((NH4)2SO4).

• Dialysis is a procedure to separate proteins from solvents

Guidelines for protein purification

• Define objectives• Define properties of target protein and

critical contaminants• Minimize the number of steps• Use a different technique at each step• Develop analytical assays

Adapted from: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC

How pure should my protein be?

Moderately high < 95%N-terminal sequencing, antigen for antibody production, NMR

High 95-99%Biochemical assays, X-ray crystallography

Extremely high > 99%Therapeutic use, in vivostudies

Required PurityApplication

Separation of proteins based on physical and chemical properties

• Solubility

• Binding interactions

• Surface-exposed hydrophobic residues

• Charged surface residues

• Isoelectric Point

• Size and shape

Basic scheme of protein purification

From: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC

Protein preparation, extraction, clarification

Cell growth, protein over-expression

Cell lysis

Removal of cell debris

Expression of Target Protein in E. coli

transformationDinI

expression

Plasmid with dinI

E. coli

Protein isolation, concentration, and stabilization

Reversible precipitation with salt or

organic molecules

Fractional precipitation of proteins

Add Precipitant,

CentrifugationChromatography

Precipitate contaminants

Precipitate protein of interest

Discard supernatant, Resuspend

protein

Discard pellet

Add Precipitant, Centrifugation,

Discard supernatant, Resuspend protein

Intermediate Purification

Liquid chromatography (lower resolution,

lower cost)

Types of liquid chromatography

Adsorption Chromatography– Proteins bind to stationary phase

– Proteins eluted by altering mobile phase

– Includes: affinity, hydrophobic interaction, ion exchange, and chromatofocusing

Solution Phase Chromatography– Proteins do not bind to stationary phase

– Progress of proteins through column impeded by matrix of stationary phase

– Includes: size exclusion chromatography (aka gel filtration)

Types of liquid chromatography

Sephacryl #, Sephadex #Same BufferSize / Shape of

ProteinPoresSolution Phase

Size Exclusion (gel filtration)

Mono-PpH gradientPoly-bufferIsoelectric PointNegatively charged

ionsAdsorptionChromatofocusing

Mono-S, Source-S, CMHigh SaltLow SaltCoulombic

InteracionsNegatively charged

ionsAdsorptionCation Exchange

Mono-Q, Source-Q, DEAEHigh SaltLow SaltCoulombic

InteracionsPositively charged

ionsAdsorptionAnion Exchange

Butyl sepharose, Octylsepharose, Phenyl

sepharoseLow SaltHigh SaltHydrophobic

EffectHydrophobic

GroupsAdsorptionHydrophobic Interaction

Metal, Ig, Hydroxyapatite,

Heparin Sepharose, Any ligand

High [Ligand]

Low [Ligand]Ligand BindingSpecific LigandAdsorptionAffinity

Names of ResinsElution With

Equilibrate WithSeparation ByResin Chemical

GroupAdsorption or SolutionType

Polishing steps

From: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC

Liquid chromatography

(higher resolution, higher cost)

Liquid chromatography techniques advantages and disadvantages

ResolutionDisadvantagesAdvantagesType of Chromatography

Low to HighLong run time

Distinct from other techniques, Can be

used analytically or for buffer exchange

Size Exclusion

HighpH gradient can be harsh for proteinHigh resolutionChromatofocusing

Medium to HighProtein solution must start at low [salt]Versatile resin choicesIon Exchange

Low to MediumRelatively low

resolution and binding capacity

Can be used directly from ammonium

sulfate precipitation

Hydrophobic Interaction

Low to MediumResins and ligands can be expensiveQuick and specificAffinity

Protein detection methods

• SDS-PAGE– Visual confirmation

• UV Spectrophotometry– Absorbance @ 280 nm– Due mostly to Trp– [Protein] calculated with Beer’s Law

• Colorimetric Techniques– Color change proportional to [protein]– Bradford, Lowry, BCA

J.S.C. Olson and John Markwell. Current Protocols in Protein Science (2007) 3.4.1-3.4.29

Final steps in purification• Check purity by detection methods

• Test for interfering contaminants– Nucleases– Proteases– Toxins

• Concentrate your protein– Precipitation– Centricons– Small column with high binding capacity

• Choose a storage buffer and storage conditions– Consider intended use of protein– Stabilizing additives– Flash freeze protein and store at -80o C

• Confirm identity of purified protein– Mass spectrometry– N-terminal sequencing– Analytical assays

Basic scheme of protein purification

Liquid chromatography

(higher resolution, higher cost)

Liquid chromatography (lower resolution,

lower cost)Reversible

precipitation with salt or

organic molecules

Cell growth, protein over-expression

Cell lysis

Removal of cell debris

Separation Processes that can be Used to Fractionate Proteins

Separation Process Basis of SeparationPrecipitation ammonium sulfate solubility

polyethyleneimine (PEI) charge, sizeisoelectric solubility, pI

Chromatography gel filtration (SEC) size, shapeion exchange (IEX) charge, charge distributionhydrophobic interaction(HIC) hydrophobicityDNA affinity DNA binding siteimmunoaffinity (IAC) specific epitopechromatofocusing pI

Electrophoresis gel electrophoresis (PAGE) charge, size, shapeisoelectric focusing (IEF) pI

Centrifugation sucrose gradient size shape, density

Ultrafiltration ultrafiltration (UF) size, shape

Protein Purification: Column Chromatography

• The expansion of the protein band in the mobile phase is caused by separation of proteins with different properties and by diffusional spreading. As the length of the column increases, the resolution of two types of protein improves.

• Rate is decreased and resolution can decline because of the diffusionalspreading

Ion-exchange Chromatography

• Cation exchangers contain negatively charged polymer

• Anion exchangers contain positively charged polymer.

• Is effected by pH

Size-Exclusion Chromatography

• Also called gel filtration: The column matrix is a cross-linked polymer with pores of selected size.

• Larger protein migrate faster than smaller ones because they are too large to enter the pores

Affinity Chromatography• Separate protein by

their binding specificities. The proteins retained on the column are those that bind specifically to a ligand cross-linked to the beads. Proteins that do not binds to ligands are washed through to column

Electrophoresis

• Separation of porteins is based on the migration of charged protein in an electric field

• The migration of a protein in a gel during electrophoresis is a function of its size and shape

µ = V/E = Z/ fµ : electrophoretic mobilityV: velocity; E: electrical potentialZ: net charge; f: frictional

coefficient

SDS-PAGE: Sodium Dodecyl Sulfate (SDS) Polyacrylamide Gel Electrophoresis

• SDS binds to most proteins probably by hydrophobic interaction. One SDS for every two AAs, Thus, each protein has a similar charge-to-mass ratio.

• Coomassie blue stains protein. Western blot

Estimating the Molecular Weight of a Protein

Isoelectric Focusing

• pI of a protein: net charge=0

• A pH gradient is established by allowing a mixture of organic acids and bases (ampholytes). Protein migrates until it reaches the pH that matches its pI

Two-Dimensional Electrophoresis

• Separates proteins of identical MW that differ in pI or proteins with similar pI but different MW.

Activity Vs. Specific Activity

• Unit: amount of enzyme causing transformation of 1 µ mole of substrate per min. at 25 oC under optimal conditions

• Activity: Total units of enzyme (U).

• Specific activity: (U/mg) of total protein

Bacterial expression vectors

Bacterial expression vectors

Mammalian expression vector