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ISOLATION, PURIFICATION AND ISOLATION, PURIFICATION AND CHARACTERIZATION OF PROTEIN CHARACTERIZATION OF PROTEIN

Isolation, Purification and Characterization of Proteins

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Page 1: Isolation, Purification and Characterization of Proteins

ISOLATION, PURIFICATION AND ISOLATION, PURIFICATION AND CHARACTERIZATION OF PROTEINCHARACTERIZATION OF PROTEIN

Page 2: Isolation, Purification and Characterization of Proteins

DIFFERENT TYPES OF PROTEIN

Intra cellular proteins:Produced inside the cellEx: Bacteria

Extra cellular proteins:Produced outside the cellEx: Monoclonal antibodies (mammalian

cells)

Page 3: Isolation, Purification and Characterization of Proteins

Objective

To Obtain maximum purity and recovery

Simplify technique selection and optimization.

Fast detection of protein activity/recovery. To minimize sample handling. Remove damaging contaminants and

enzymes early. The technique must be cost effective and

also not time consuming.

Page 4: Isolation, Purification and Characterization of Proteins

Sample and target protein properties Influence of purification Stratergy

Temperature Stability Need to word rapidly at lower temperature

pH Stability Selection of buffers

Protease Sensitivity Need for fast removal of proteases

Sensitivity to metal ions EDTA

Molecular weight Selection of Gel filtration media

Charge properties Ion exchange conditions

Bio specific affinity Ligand or affinity medium

PARAMETERS TO BE LOOKED UP

Page 5: Isolation, Purification and Characterization of Proteins

Protein from cells or tissue

Microbial cells or tissue

Break cells, tissue, or organ

Blender, homogenizer, sonication,pressure,osmotic shock

Pellet with intact cells, organelles, membranes and membrane proteins

Supernatant withSoluble protein

Separated by centrifugation and filtration

ChromatographyCharacterization

Concentration of The product

STEPS INVOLVED IN EXTRACTION OF STEPS INVOLVED IN EXTRACTION OF PROTEINSPROTEINS

Choose a suitable clone INOCULATION in fermenter

Extraction

INTRACELLULAR

EXTRACELLULARDesiredcells

Page 6: Isolation, Purification and Characterization of Proteins

Steps for complete extraction of intracellular proteinSteps for complete extraction of intracellular protein

Page 7: Isolation, Purification and Characterization of Proteins

ISOLATION• Extraction from the cell body.• Mixture of components.• Non – protein materials.

CLARIFICATION / PURIFICATION• Remove the cell debris• Chromatography

CONCENTRATION• Chromatography• Concentrating the protein

CHARACTERIZATION• Determining the various parameters

STEPS INVOLVED:

Page 8: Isolation, Purification and Characterization of Proteins

Isolation Methods Osmotic Shock Homogenizers Grinding The Parr Bomb Extrusion under high pressure Sonication Enzyme digestion

Page 9: Isolation, Purification and Characterization of Proteins

Osmotic Shock

Mechanical means of disrupting cells with buffer of low osmotic pressure

Buffer flows in the cells and lysis of cells happens with the release of the desired protein.Ex: n - butanol

Page 10: Isolation, Purification and Characterization of Proteins

Homogenizers

Pestle homogenizers Virtis homogenizers Polytron homogenizers

Generally disrupts the cell but not the organelles

Page 11: Isolation, Purification and Characterization of Proteins

Grinding & Parr bomb

Edmund buhler disintegraterBacterial cells are vibrated with

glass beads in a jacketed container.

Sample is subjected to nitrogen which penetrates in the cell when pressure is released bubbles come and disrupts the cells.

Page 12: Isolation, Purification and Characterization of Proteins

Extrusion under high pressure

Cells are broken through passing a narrow orifice

Laminar air flow shears the cells and passes thro’ a needle valve

Page 13: Isolation, Purification and Characterization of Proteins

Sonication

High frequency sound waves are passed

Thro’ a method of ‘micro – cavitations’ ie production of low transient pressure by which disruption of cells happen.

Page 14: Isolation, Purification and Characterization of Proteins

Enzyme digestion

Using an enzyme to digest the cell walls so that the cell breaks and opens up with cell organelles

Ex: Bacterial cells – Lysozyme Fungal cells - Chitinases Plant cells - Cellulases

Page 15: Isolation, Purification and Characterization of Proteins

Extraction Process Typical Conditions Protein Source Comment

Osmotic Shock 2 volumes of water to 1 volume packed

intracellular proteins Lower product release with little protease release

Enzyme digestion Lysozyme 0.2mg/ml 37’c for 15min

Bacteria, intracellular proteins

Lab scale only, often combined with mechanical digestion

Homogenization Follow equipment procedure

Liver tissue, muscle tissue, cell suspension

Large scale only

Ultra sonication or bead milling

Follow equipment procedure

Cell suspensions and intracellular proteins

Small scale only

French press Follow equipment procedure

Bacteria and plant cells

-

Fractional precipitation

Follow equipment procedure

Monoclonal antibodies, cell lysates

Precipitates must be resolubilized

Page 16: Isolation, Purification and Characterization of Proteins

Clarification

Filtration

Density gradient centrifugation

Chromatography

Page 17: Isolation, Purification and Characterization of Proteins

Centrifugation

Density gradient centrifugationMechanism – high density cell debris settles down and desired proteins are retained in the supernatant

By this method only the cell debris are only removed but contaminants like HCP’s, HCDNA’s, particles and other proteins are not removed.

Page 18: Isolation, Purification and Characterization of Proteins

Filtration

Filtration is depending on the pore size.Mainly the particles are removed in

filtration techniques and it makes the sample feasible to use in the next step.

Page 19: Isolation, Purification and Characterization of Proteins

Chromatography

Charge Ion Exchange

Size Gel Filtration

Hydrophobicity Hydrophobic interaction/reverse phase

Bio recognition (ligand specificity) Affinity

Charge, ligand specificity, Hydrophobicity

EBA – expanded bed absorption

Page 20: Isolation, Purification and Characterization of Proteins

Buffer Components Typical Conditions for use

Purpose

Tris 20mM, pH = 7.4 Maintain pH, minimize acidification caused by lysosomal disruption

NaCl 100mM Maintain ionic strength

EDTA 10mM Reduce oxidation damage, Chelate metal ions

Sucrose or glucose 25mM Stabilizes lysosomal membranes, reduce protease release

COMMON BUFFERS USED

Page 21: Isolation, Purification and Characterization of Proteins

Concentration

Freeze dryingDialysisBy salting outTPP – Three phase partitioningTFF

Specific gravity increases

Page 22: Isolation, Purification and Characterization of Proteins

Freeze Drying

Long time storageRemoval of water from the sample by

sublimation.

This method might destroy activity of some protein and hence forth sample should be checked before introducing.

Page 23: Isolation, Purification and Characterization of Proteins

Dialysis

Diffusion of solutes thro’ a semi permeable membrane

Donnan membrane Effect

Counter current dialysis

Page 24: Isolation, Purification and Characterization of Proteins

Ultra filtration & Salting out

Desalting or buffer exchange Size fractionation

Using Ammonium Sulfate Three phase partitioning

Page 25: Isolation, Purification and Characterization of Proteins

Precipitation

With Polyethylene glycolWith organic solventsDye precipitation

Page 26: Isolation, Purification and Characterization of Proteins

TFF

Tangential Flow Filtration

Used to concentrate protein with the use of a membrane cassette

Can concentrate protein with very minimum product loss.

Page 27: Isolation, Purification and Characterization of Proteins

After the isolation and purification of proteins, they must be stored in suitable conditions for a longer time

Should be devoid of aggregation and other problems

SAMPLE STORAGE

Page 28: Isolation, Purification and Characterization of Proteins

Quantification

Amino acid analysis

Disulphide Linkage Determination

PM Spectrometry

Antibody Characterization

Purity assessment

SDS PAGE

IEF

RP HPLC

Peptide MappingAdvanced Characterization Endotoxins and Host Cell Contamination

ELISA

HPLC

LAL Assay

PM by LC/MS

Aggregate Sequence Analysis

Amino acid Analysis

Higher order structure

Protein folding Determination of Extinction coefficientAmino-acid

analysis

Colorimetric Protein assay

Dynamic Light Scattering

Size Exclusion chromatography

Mass Spec

Edman Sequencing

Precolumn Derivitization and HPLC

Hydrolysis

X ray diffraction

NMR

Spectroscopy

NMR

Functional Assay

UV spectroscopy

METHODS FOR PROTEIN CHARACTERIZATIONMETHODS FOR PROTEIN CHARACTERIZATION

Page 29: Isolation, Purification and Characterization of Proteins

THANK YOUTHANK YOU

Courtesy: Courtesy: Isolation of Proteins By Clive DenninsonIsolation of Proteins By Clive DenninsonProtein Purification, Principle and Practice, R.K.ScopesProtein Purification, Principle and Practice, R.K.ScopesProtein Purification, Amersham BiosciencesProtein Purification, Amersham BiosciencesIntro to TFF, Pall BiosciencesIntro to TFF, Pall Biosciences

By

Anand.D