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قسم الكيمياء الحيوية

Protein PurificationProtein Purification and Characterization

دولت على سالمه.دالحيوية استاذ الكيمياء٢٠١٥-٢٠١٤

المحاضرة الخامسة 512: الرمز الكودي

The students should be learning about Homogenization centrifugation g Dialysis Precipitation

Chromatography Chromatography Ion-exchange chromatography Affinity chromatography Affinity chromatography

Gel filtration

Reversed phase high performance liquid chromatography

Electrophoresis Analysis SDS-PAGE Isoelectric focusing 2D electrophoresis

M t Mass spectroscopy

Mechanical Shearing - Warring blender, glass or plastic pestle like h ihomogenizer

Freeze Thaw - cycles under hypnotic conditions, ice crystals disrupts membranesdisrupts membranes

Enzymatic degradationEnzymatic degradation of the cell membrane of the cell membrane --l f b i l il f b i l imostly for bacterial preparationmostly for bacterial preparation

SonicationSonication -- high energy sounds to disrupt high energy sounds to disrupt membranemembranemembranemembrane

DetergentDetergent disruption of disruption of membranemembrane

Physical Characteristics Distinguishing Proteins

• Because of the differences in size andBecause of the differences in size and shape, proteins will sediment gradually under the centrifugal force until theunder the centrifugal force until the sedimentation force and buoyant forcereach the balance.

• The sedimentation behavior is described• The sedimentation behavior is described in sedimentation coefficient (S) which is

ti l t th l l i htproportional to the molecular weight.

• One of the most used methods in biochemistryy

• Uses increasing g forces to yield a pellet and a supernatant

• Subcellular centrifugation a way to• Subcellular centrifugation - a way to separate the cell contents based on density mass, shape and the density of the solution

• Cytosol - not an organelle but a result of centrifugation

600 g 15 000 g 105 000 g

Cytosolicfraction

600 gx 10 min

15,000 gx 5 min

105,000 gx 60 min

Homogenate Nuclearfraction

Mitochondrialfraction

Microsomalfraction

• Proteins, as macromolecules, cannot pass through the semipermeable membrane containing pores of smaller than protein dimension, thus large

t i d ll l l b t dproteins and small molecules can be separated.

• Dialysis can be used for protein purification, desalting, and condensation. y p p , g,

• Adding a large quantity of salts such as Ammonia sulfateAdding a large quantity of salts, such as Ammonia sulfate, into the protein solution will neutralize the surface charges and the destruct the hydration shell of proteins, causing them to precipitate.

• Acetone has the similar function.

• An efficient way to concentrate proteins.

Each protein has a different solubility so this is a method solubility so this is a method to isolate groups of protein

Salting out• One of the most commonly used methods for purification• Proteins can be precipitated by salting-out b/c an excess of saltProteins can be precipitated by salting-out b/c an excess of salt

in solution competes with the protein for the water molecules, leaving the protein with no bulk water available to dissolve in

Takes away water by interacting with it, makes protein less soluble because hydrophobic interactions among proteins increases

– Thus, proteins are ‘salted-out’ of water by salt– By progressively changing salt conc., the protein of interest

can be partially purified

centrifugationg

4.Column Chromatography4.Column Chromatography

When a protein solutionWhen a protein solution (called as mobile phase) passes through a stationarypasses through a stationary phase, proteins can interact with the stationary phasewith the stationary phase due to the differences in size, charge, and affinity,size, charge, and affinity, making the different proteins flow through the stationaryflow through the stationary phase at different speeds.

Elutionbuffer

Protein mixture

Solidphasecapableofreactingwithproteinsproteinsto beseparated

Protein 2Protein 1

OD

280n

m

Elution volume

Type of chromatographyType of chromatography

Ion e change based on the ionic interactions• Ion exchange: based on the ionic interactions

• Affinity: based on the binding strengths• Affinity: based on the binding strengths

• Filtration: based on the protein sizesp

• Hydrophobicity: based on the hydrophobic forces

Ion exchange chromatography

M t i l i th ti ll d d i ti f

Ion-exchange chromatography

Material is synthetically prepared derivatives ofcellulose:

diethylaminoethylcellulose (DEAE-cellulose)diethylaminoethylcellulose (DEAE-cellulose)carboxymethylcellulose (CM-cellulose)

• DEAE-cellulose contains (+) charges (pH 7.0)anion exchanger

• CM-cellulose contains (-) charges (pH 7.0)cathion exchangercathion exchanger

•Is a separation mode in which, if the solutes and stationary phase have the i h h d h h b l i i iopposite charge, they are attracted to each other by electrostatic interaction,

and the solutes are consequently retained. •Ion exchange chromatography can be divided into two types according to the g g p y yp gcharge on the target solutes:

••anion anion exchangeexchange chromatographychromatography••and cation exchange chromatographyand cation exchange chromatography.

N+

RRAnion exchange

R

++SO3

- ++

+++

++

+++

Electrostatic interaction

Cation exchange

Electrostatic interaction(Coulomb force)

ti f t i b d t h f - separation of proteins based on net charge of protein - exchange of ions for proteins

Ion-exchange chromatographyIon exchange chromatographyanion exchange chromatographyanion exchange chromatography

More negatively chargedproteins bind to the solidphase tightly, and strongerl ti b ff i d d t

－＝

elution buffer is needed toelute them out the column

＋

＋

＋＋

＋

＋

＋

＋＋

＋

－

－

－

＝

＝＝

＝

＝

＝

＋

＋

＋＋

＋

＋－

－－

＝

＝

＝

＝

＝－

－

－Ionic exchangecolumn withpositive chargeLess negatively charged

proteins bind to the solidproteins bind to the solidphase loosely, and weakelution buffer can be used toelute them out the column

• In an amino acid analyzer, the hydrolysate passesthe hydrolysate passes through an ionexchange column. Th l ti i f The solution emerging from the column is treated with ninhydrin, and its absorbance is recorded as a function of time.

Each amino acid is identified by the retention time required to pass through the columnthrough the column.

44..22.Affinity chromatography.Affinity chromatographypurification based on a natural interactions for a protein and a purification based on a natural interactions for a protein and a p pp p

substrate or chemical group (ligand)substrate or chemical group (ligand)–– only proteins which recognize the molecule on the stationary only proteins which recognize the molecule on the stationary

phase will bindphase will bindphase will bindphase will bind–– Elute by competition with the bound ligandElute by competition with the bound ligand–– generally a good method but it doesn’t always work generally a good method but it doesn’t always work -- Some Some g y g yg y g y

nonnon--specific interactions can occurspecific interactions can occur–– Spacer arm may be needed to make the Spacer arm may be needed to make the

d il bl t th t id il bl t th t icompound available to the proteincompound available to the protein

44..33.Size exclusion (SEC) or gel filtration .Size exclusion (SEC) or gel filtration chromatographychromatographygg

Media (solid phase) is a defined pore sizes in polymer beads, Media (solid phase) is a defined pore sizes in polymer beads, large molecules “go around” small molecules “go through and large molecules “go around” small molecules “go through and

around the beads”around the beads”Smaller sized proteins are retained and come out lastSmaller sized proteins are retained and come out lastAlso used to Also used to determine the molecular weight of a protein determine the molecular weight of a protein -- use use

protein protein standards with known molecular weights, prepare a standards with known molecular weights, prepare a standard curve of these known proteins and compare the elution standard curve of these known proteins and compare the elution standard curve of these known proteins and compare the elution standard curve of these known proteins and compare the elution volumes of the volumes of the knownsknowns to the unknownsto the unknowns

Gel filtrationS m a l l p r o t e i n s c a ne n t e r t h e p o r o u sb e a d s , a n d h a v e a

Gel filtration

b e a d s , a n d h a v e al o n g e r s t a t i o n a r yt i m e

L i hP o r o u s b e a d s

L a r g e p r o t e i n s t h a ta r e u n a b l e t o e n t e rt h e p o r o u s b e a d s w i l lp a s s b y a n d f l o w o u t

a l l o w t h e s m a l lp r o t e i n s e n t e r

p yd i r e c t l y

4.4.Reversed phase high performance liquid chromatographychromatography

Reversed Phase Chromatography - Proteins with exposed hydrophobic region (red) will be able to bind to the immobilized hydrocarbon stationary phase; the rest simply wash off

R Ph h t h i ti b dReverse Phase chromatography is a separation based on the solubility of the protein .

Monitoring Progress ofMonitoring Progress of Purification ProtocolPurification Protocol

• Total protein (mg)Total protein (mg)– Quantity of protein present in fraction

• Total activity (units of activity)• Total activity (units of activity)– Use a portion of sample to determine activity.

– Multiply activity by total volume to determine total activityactivity.

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• Specific activity (units of activity/mg)T t l ti itTotal activityTotal protein

S.A. =

• % yield: measure of activity retained after each step in procedure.

% i ld Total activity at particular step% yield =Total activity at particular stepTotal activity of initial extract

24


• Purification level: Measure of increase in purity of protein throughout procedureof protein throughout procedure.

Purification = Specific activity at particular stepSpecific activity of initial extractp y

25

Edited by Janway Chen

Monitoring Progress of Purification ProtocolPurification Protocol

Step Total protein (mg)

Total activity (units)

Specific activity

(units/mg)Yield (%) Purification

level

Initial extract 15,000 150,000 10 100 1

(NH4)2SO4(NH4)2SO4precipitation 4,600 138,000 30 92 3

Ion-exchange 1,278 115,500 90 77 9exchange , ,

Size exclusion 68.6 75,000 1,100 50 110

Affinity column 1.75 52,500 30,000 35 3,000

(Berg, Tymoczko, & Stryer. (2002) Biochemistry, 5th ed. W.H. Freeman & Co., New York, NY, p. 86)

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Setting up a purification Tableg p pStep Activity

(µmol/min/ml)Proteinconc.

(mg/ml)

Totalvolume

(ml)

Totalprotein

(mg)

Total Activity(µmol/min)

Specific activity(µmol/min/mg)

Fold Purification

Yield (%)

( g ) ( ) ( g)

Given Given Given Given Given To be determined

TBD TBD TBD

Total Enzyme Activity: the activity per ml of enzyme solutionTotal Enzyme Activity: the activity per ml of enzyme solution multiplied by the total volume of that fraction.

T t l P t i th t i t ti l f l ti lti li dTotal Protein: the protein concentration per ml of solution multiplied by the total volume of that fraction.

Specific activity: Total Activity/Total Protein or Activity/Protein concentration

Fold-purification: (Specific activity at a given step)/(Specific activity of starting sample)

Yield = (Total activity at a given step) / (Total activity of starting sample)*100

From LehningerPrinciples of Biochemistry

ElectrophoresisElectrophoresis

-- The transport of particles by an The transport of particles by an electrical field through a solid mediaelectrical field through a solid mediaelectrical field through a solid mediaelectrical field through a solid media

-- a good method for determining the a good method for determining the purity of a protein and analyze a mixture ofpurity of a protein and analyze a mixture ofpurity of a protein and analyze a mixture of purity of a protein and analyze a mixture of proteinsproteins

--Separation of charged compounds Separation of charged compounds p g pp g pbased on an applied electrical field, net based on an applied electrical field, net charge and frictional coefficient (mass and charge and frictional coefficient (mass and h f l l )h f l l )shape of molecule)shape of molecule)

Similar to DNA gelsSimilar to DNA gelsproteins and very small DNA proteins and very small DNA (oligonucleotides) use acrylamide(oligonucleotides) use acrylamide

1. Native PAGE

2. Native Gradient PAGE

3. Urea PAGE3. Urea PAGE

4. SDS PAGE

5 SDS Gradient PAGE5. SDS Gradient PAGE

6. IEF

7 2D PAGE7. 2D PAGE

8. Western Blot

DefinitionElectrophoresis is the migration of charged particles such as proteins dissolved in a buffer on, or in, a supporting medium when an electric current is passed through them

1-Sodium dodecyl sulfate polyacrylamide y p y ygel electrophoresis (SDS-PAGE)

• Sodium dodecyl sulfate is a kind of detergent to denature the proteins

• Anionic SDS bind to protein in the ratio ofAnionic SDS bind to protein in the ratio of 1 SDS per 2 AAs.

• The protein-SDS complex is roughly charged proportional to the mass.

• The smaller the protein, the faster the moving speed in the electric fieldmoving speed in the electric field.

• The gel polymer material for protein discrimination is composed ofdiscrimination is composed of methylenebisacrylamide and acrylamide.

• The pore size can be controlled by changing the concentration of cross-linkingchanging the concentration of cross linking reagent.

• The gel with different concentration of cross-linking reagent can be used forcross linking reagent can be used for different size proteins.

٣٦٣٦

2-Isoelectric Focusing (IEF)g ( ) Isoelectric focusing separates proteins according to their

chargecharge. PAGE gel is saturated with ampholytes, a mixture of

polyanionic and polycationic molecules. I l t i fi ld h l t t d f di t In electric field ampholytes separate and form a gradient based on their net charge

Ampholyte gradient establishes a pH gradientProteins migrate through the gradient until they reach their pI the pH at which the net charge is zero

I CathodeAnode

pI, the pH at which the net charge is zero

pI

+

+ --- - - -

+ + + + + ++-

CathodeAnode

٣٧

+ + + + + + ++pH 4 pH 7

Electrophoresis3.Two-dimensional gel electrophoresis

• Here, the proteins are FIRST Here, the proteins are FIRST separated according to their pIby IEF.

• THEN the strip of IEF gel is• THEN, the strip of IEF gel is placed on top of a PAGE-SDS gel: the proteins will then be separated according to their Mr;separated according to their Mr;

• Allows a much higher resolution of the proteins

i h i fpresent in the mixture of interest;

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2D PAGE

٣٩

Antibodies in specific Antibodies in specific l il ianalysisanalysis

•• ELISA (Enzyme Linked ImmunoAssay)ELISA (Enzyme Linked ImmunoAssay)-- most sensitive detection most sensitive detection methods for antibodies (aids test) proteins peptides and other methods for antibodies (aids test) proteins peptides and other methods for antibodies (aids test), proteins, peptides and other methods for antibodies (aids test), proteins, peptides and other substances (drug testing)substances (drug testing)

Unreacted binding sites are Unreacted binding sites are 22

Protein of interest is Protein of interest is

Covered with a nonCovered with a non--reactive reactive proteinprotein

11Plastic Dish

Protein of interest is Protein of interest is Bound to plasticBound to plastic



Unreacted binding sites are Unreacted binding sites are 22

Primary AntibodyPrimary AntibodyRecognizes AntigenRecognizes Antigen



1133

Plastic DishProtein of interest is Protein of interest is Bound to plasticBound to plastic



Secondary AntibodySecondary AntibodyConjugated to an enzymeConjugated to an enzyme Unreacted binding sites are Unreacted binding sites are 22

44




1133


Antibodies in specific analysisAntibodies in specific analysisAntibodies in specific analysisAntibodies in specific analysis•• 11. . ELISA (Enzyme Linked ImmunoAssay)ELISA (Enzyme Linked ImmunoAssay)-- most sensitive detection most sensitive detection

methods for antibodies (aids test) proteins peptides and other methods for antibodies (aids test) proteins peptides and other methods for antibodies (aids test), proteins, peptides and other methods for antibodies (aids test), proteins, peptides and other substances (drug testing)substances (drug testing)

Enzyme reacts with Enzyme reacts with substrate producing substrate producing colored productcolored product

55

Secondary AntibodySecondary AntibodyConjugated to an enzymeConjugated to an enzyme Unreacted binding sites are Unreacted binding sites are

colored productcolored product

22

44




1133


2.Western blot –• good for mixtures of proteins,

identifying size and characteristics– transfer proteins form SDS PAGE to

paper for antibody analysis.– Primary antibody recognizes protein

antigen– a secondary antibody recognizes the

Fc region and is conjugated to a second molecule to act as a signalsecond molecule to act as a signal

• Newly developed approach applied to biology and medicine areasbiology and medicine areas

• Revolutionized bioanalytical technique• Offering a fast, high accuracy, and high

throughput determination for analyzingthroughput determination for analyzing peptides and proteins.

Overview of steps involved in proteomic analysis

Step 1: Sample prep

Step 2: Separation

Matrix-aided ionizationMatrix aided ionization

1 D it l1. Deposit samples on a plate.

2. Introduce a beam of laser.

3. Ionize samples. 4 Analyze ionized4. Analyze ionized

molecules. 5 Determine the AA5. Determine the AA

sequence.

4. Structure Determination4. Structure Determination

• X-ray crystallography • Nuclear magnetic resonanceNuclear magnetic resonance

spectroscopy

ProteomicsProteomics

a comprehensive knowledge about all the proteins of a cellabout all the proteins of a cell at a specific given time.

THANK YOUTHANK YOUTHANK YOU…..THANK YOU…..

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