protein purification hjp
TRANSCRIPT
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