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ISOLATION, PURIFICATION AND ISOLATION, PURIFICATION AND CHARACTERIZATION OF PROTEINCHARACTERIZATION OF PROTEIN
DIFFERENT TYPES OF PROTEIN
Intra cellular proteins:Produced inside the cellEx: Bacteria
Extra cellular proteins:Produced outside the cellEx: Monoclonal antibodies (mammalian
cells)
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.
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
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
Steps for complete extraction of intracellular proteinSteps for complete extraction of intracellular protein
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:
Isolation Methods Osmotic Shock Homogenizers Grinding The Parr Bomb Extrusion under high pressure Sonication Enzyme digestion
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
Homogenizers
Pestle homogenizers Virtis homogenizers Polytron homogenizers
Generally disrupts the cell but not the organelles
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.
Extrusion under high pressure
Cells are broken through passing a narrow orifice
Laminar air flow shears the cells and passes thro’ a needle valve
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.
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
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
Clarification
Filtration
Density gradient centrifugation
Chromatography
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.
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.
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
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
Concentration
Freeze dryingDialysisBy salting outTPP – Three phase partitioningTFF
Specific gravity increases
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.
Dialysis
Diffusion of solutes thro’ a semi permeable membrane
Donnan membrane Effect
Counter current dialysis
Ultra filtration & Salting out
Desalting or buffer exchange Size fractionation
Using Ammonium Sulfate Three phase partitioning
Precipitation
With Polyethylene glycolWith organic solventsDye precipitation
TFF
Tangential Flow Filtration
Used to concentrate protein with the use of a membrane cassette
Can concentrate protein with very minimum product loss.
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
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
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