Isolation, Purification And Characterization Of Proteins

ISOLATION, PURIFICATION AND CHARACTERIZATION OF PROTEIN

DIFFERENT TYPES OF PROTEIN Intra cellular proteins: Produced inside the cell Ex: Bacteria Extra cellular proteins: Produced outside the cell Ex: 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.

PARAMETERS TO BE LOOKED UP 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

STEPS INVOLVED IN EXTRACTION OF PROTEINS Choose a suitable clone

Desired cells

INOCULATION in fermenter

EXTRACELLULAR Extraction

INTRACELLULAR Protein from cells or tissue Supernatant with Soluble protein

Break cells, tissue, or organ

Microbial cells or tissue

Blender, homogenizer, sonication, pressure, osmotic shock

Characterization

Pellet with intact cells, organelles, membranes and membrane proteins

Chromatography

Concentration of The product

Separated by centrifugation and filtration

Steps for complete extraction of intracellular protein

STEPS INVOLVED: 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

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 disintegrater Bacterial 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 centrifugation Mechanism – 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

COMMON BUFFERS USED 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

Concentration Freeze drying Dialysis By salting out TPP – Three phase partitioning TFF Specific gravity increases

Freeze Drying  Long

time storage  Removal 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 glycol  With organic solvents  Dye precipitation

TFF  Tangential

Flow

Filtration  Used to concentrate protein with the use of a membrane cassette  Can concentrate protein with very minimum product loss.

SAMPLE STORAGE 



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

METHODS FOR PROTEIN CHARACTERIZATION Antibody Characterization

Advanced Characterization

Purity assessment

Endotoxins and Host Cell Contamination Peptide Mapping PM by ELISA LC/MS

SDS PAGE

LAL Assay

RP HPLC

Quantification

Disulphide Linkage Determination

HPLC

IEF

PM Spectrometry

Aggregate

Amino-acid analysis

Sequence Analysis

Amino acid Analysis

Dynamic Light Scattering

Colorimetric Protein assay Size Exclusion chromatography

Higher order Protein folding structure

Hydrolysis

Edman Sequencing

Precolumn Derivitization and HPLC

NMR

Determination of Extinction coefficient

NMR

X ray diffraction

Amino acid analysis

Spectroscopy UV spectroscopy Functional Assay

Mass Spec

THANK YOU Courtesy:

Isolation of Proteins By Clive Denninson Protein Purification, Principle and Practice, R.K.Scopes Protein Purification, Amersham Biosciences Intro to TFF, Pall Biosciences

By Anand.D