isolation, purification and characterization of proteins
TRANSCRIPT
![Page 1: Isolation, Purification and Characterization of Proteins](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/1.jpg)
ISOLATION, PURIFICATION AND ISOLATION, PURIFICATION AND CHARACTERIZATION OF PROTEINCHARACTERIZATION OF PROTEIN
![Page 2: Isolation, Purification and Characterization of Proteins](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/2.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/3.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/4.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/5.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/6.jpg)
Steps for complete extraction of intracellular proteinSteps for complete extraction of intracellular protein
![Page 7: Isolation, Purification and Characterization of Proteins](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/7.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/8.jpg)
Isolation Methods Osmotic Shock Homogenizers Grinding The Parr Bomb Extrusion under high pressure Sonication Enzyme digestion
![Page 9: Isolation, Purification and Characterization of Proteins](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/9.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/10.jpg)
Homogenizers
Pestle homogenizers Virtis homogenizers Polytron homogenizers
Generally disrupts the cell but not the organelles
![Page 11: Isolation, Purification and Characterization of Proteins](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/11.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/12.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/13.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/14.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/15.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/16.jpg)
Clarification
Filtration
Density gradient centrifugation
Chromatography
![Page 17: Isolation, Purification and Characterization of Proteins](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/17.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/18.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/19.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/20.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/21.jpg)
Concentration
Freeze dryingDialysisBy salting outTPP – Three phase partitioningTFF
Specific gravity increases
![Page 22: Isolation, Purification and Characterization of Proteins](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/22.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/23.jpg)
Dialysis
Diffusion of solutes thro’ a semi permeable membrane
Donnan membrane Effect
Counter current dialysis
![Page 24: Isolation, Purification and Characterization of Proteins](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/24.jpg)
Ultra filtration & Salting out
Desalting or buffer exchange Size fractionation
Using Ammonium Sulfate Three phase partitioning
![Page 25: Isolation, Purification and Characterization of Proteins](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/25.jpg)
Precipitation
With Polyethylene glycolWith organic solventsDye precipitation
![Page 26: Isolation, Purification and Characterization of Proteins](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/26.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/27.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/28.jpg)
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](https://reader033.vdocument.in/reader033/viewer/2022061118/5469f741af79592a298b4d1e/html5/thumbnails/29.jpg)
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