Download - 3 Teknik Pemurnian Protein Muthi
III. PROTEIN PURIFICATION
BIOKIMIA
Laboratorium Kimia MedisinalBagian Kimia FarmasiFakultas Farmasi UGM
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Cakupan
• Sifat protein• Metode pemisahan protein:
FraksinasiPengendapanKromatografi
• Penentuan bobot molekul protein.
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Methods in Protein Chemistry
These are methods used in isolation, purification, detection, degradation, analysis and synthesis of proteins.
As one would expect, most of these involve aqueous media and require a knowledge of pH, pKas, and charge on a peptide at various pH values.
Proteome: defines the compete functional information about a group of proteins thatwork together as a functional unit.
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Protein Concentration from Absorbance
Beer’s LawA = cl
The proteinabsorbancemeasured at280 nm is due to Tyr & Trp
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Protein Purification Starting Material
Start with a source very rich in protein: Organism, tissue, cell type
Can you isolate a particular organelle as a starting purification step?
PEMURNIAN
MEMECAH SEL
PEMURNIAN
FRAKSINASI
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How
to S
epara
te T
hese
Ob
jects
1 2 3
9 10 11 12
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4 85
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4
5
8
wood stone cotton wood wood cotton stone wood stone cotton stone cotton
cotton
wood
stone
ShapeSizeDensity
Shape
Density
Size
Sieving different sizes Different sedimentationDifferent rolling speed
4 6 7 85
1 3 4 6 7 8 9 10 11 122 5
Juang RH (2004) BCbasics
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Basic Principles of Protein Purification
Ammonium sulfate fractionation
Cell OrganelleHomogenization
MacromoleculeNucleic
acid Carbohydrate (Lipid)
Size Charge Polarity Affinity
Small molecule Cell DebrisProtein
Amino acid, Sugar,
Nucleotides, etc
Gel filtration,SDS-PAGE,Ultrafiltration
Ion exchange,Chromatofocusing,
Disc-PAGE,Isoelectric focusing
Reverse phasechromatography,
HIC,Salting-out
Affinitychromatography,Hydroxyapatite
Juang RH (2004) BCbasics
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Begin with intact tissue
• Disrupt– Blender, homoginizer
• Remove debris– Centrifugation
• Precipitate/concentrate– Ammonium sulfate
• Purify– Chromatography
• Analyze– Activity, molecular weight
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Cell disruption (for intracellular enzymes)
• Sonication– Use of high frequency sound waves to disrupt cell walls and
membranes• Can be used as continuous lysis method• Better suited to small (lab-scale) operations• Can damage sensitive proteins
• Pressure cells– Apply apply high pressure to cells; cells fracture as pressure is
abruptly released• Readily adapted to large-scale and continuous operations• Industry standard (Manton-Gaulin cell disruptor)
• Enzymic lysis– Certain enzymes lyse cell walls
• Lysozyme for bacteria; chitinase for fungi• Only useful on small laboratory scale
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Downstream process depends on product use
1. Enzyme preparations for animal feed supplementation (e.g., phytase) are not purified
2. Enzymes for industrial use may be partially purified (e.g., amylase for starch industry)
3. Enzymes for analytical use (e.g., glucose oxidase) and pharmaceutical proteins (e.g., TPA) are very highly purified
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Fermentation
Culture supernatant
Centrifugation to remove cells
Liquid preparation to animal feed
market
Fermentation
Culture supernatant
Fermentation
Cell pellet
Intracellular fraction
Animal feed enzyme Analytical enzyme Therapeutic protein
Centrifugation to remove cells
Centrifugation to remove medium
Proteinprecipitation
Celllysis Centrifugation
Protein fraction
Proteinprecipitation
Protein fraction
1 or 2 purificationsteps
Semi-purifiedprotein 3-4 purification
steps
Homogeneousprotein
Sterile bottling
To pharmaceuticals market
LyophilisationBottling
To chemicals market
Centrifugation
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Separation of a cell homogenate.
Metode umum pemurnian
• Precipitation : temperature, pH, Salting out– Different proteins precipitate under
different solution conditions- can use soluble or insoluble fractions
• Chromatography: fractionation of contents in solution based on selection by a stationary phase
• Ultracentrifugation• Vacum dialysis• Freeze drying
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Solubility of Proteins
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Salting in: When proteins are placed in an aqueous solution, the only ionic species in solution are the other protein molecules. Water, although polar, is only slightly ionized so the proteins tend to aggregate based on ionic interactions that form between themselves. The interactions between protein molecules are more favorable than interactions between water and a protein.
At low salt concentration (NaCl), other ionic species are now present to compete with the ionic protein:protein interactions. As a result, the ionic interactions between proteins break up and the proteins dissolve. Both the small ions (from NaCl) and the proteins are solvated by water.
Solubility of Proteins
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Salting out: At high salt concentration (typically with (NH4)2SO4 or Na2SO4), water molecules are more strongly attracted to these small ions (especially multivalent ions) than to the large protein molecules. The proteins are left then to seek whatever favorable interactions exist and these are the protein:protein associations which result in aggregation and precipitation.
Isoelectric precipitation: At the pI there is zero net charge on a protein. At a pH away from the pI, each protein molecule bears an identical charge (either + or - depending on the pH) resulting in repulsion between molecules. At the pI, no repulsion occurs, and the proteins will aggregate and precipitate.
Salting in / Salting out
• Salting IN• At low concentrations,
added salt usually increases the solubility of charged macromolecules because the salt screens out charge-charge interactions.
• So low [salt] prevents aggregation and therefore precipitation or “crashing.”
• Salting OUT• At high concentrations
added salt lowers the solubility of macromolecules because it competes for the solvent (H2O) needed to solvate the macromolecules.
• So high [salt] removes the solvation sphere from the protein molecules and they come out of solution.
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“Salting out”
“Salting in”
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Kosmotrope vs. Chaotrope
• Ammonium Sulfate• Increasing conc
causes proteins to precipitate stably.
• Kosmotropic ion = stabilizing ion.
• Urea• Increasing conc
denatures proteins; when they finally do precipitate, it is random and aggregated.
• Chaotropic ion = denaturing ion.
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Dialysis
• Passage of solutes through a semi-permeable membrane.
• Pores in the dialysis membrane are of a certain size.
• Protein stays in; water, salts, protein fragments, and other molecules smaller than the pore size pass through.
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Dialysis is a form of molecular filtration• Dialysis is a process that separates molecules according to
size through the use of semipermeable membranes containing pores of less than macromolecular dimensions.
Cellophane (cellulose acetate) is the most commonly used dialysis material.
Dialysis is routinely used to change the buffer in which macromolecules are dissolved.
Dialysis can be used to concentrate a macromolecular solution by packing a filled dialysis bag in a polymeric dessicent, such as polyethylene glycol, which cannot penetrate the membrane. Concentration is effected as water diffuses across the membrane to be absorbed by the polymer.
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Dialysis
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Separation ofvery large from very small molecules is based on anattempt to equilibrateconcentration.
Osmotic pressure
Chromatography
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1. Size- sieve effect, small molecules faster
2. Ion exchange- charge attraction at protein surfaceChoose “+” stationary phase for proteins with
more “-” chargeFirst bind everything, then elute with salt
3. Hidrophobicity- Reversed Phase HPLC
4. Affinity chromatographyAntibody, binding protein Inserted tag (e.g. 6-His)
Gel Filtration Chromatography
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Gel Filtration – size separation
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Principles of gel chromatography (con’d)
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Gel Filtration Elution Volumes as a Function of Molecular Weight
Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984. 29
Gel permeation chromatography (GPC)
• Known as ‘size exclusion chromatography’ and ‘gel filtration chromatography’
• Separates molecules on the basis of molecular size
• Separation is based on the use of a porous matrix. Small molecules penetrate into the matrix more, and their path length of elution is longer.
• Large molecules appear first, smaller molecules later30
GPC in operation
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Large protein Small protein
Short path length Longer path length
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Gel Filtration (GF) Chromatography
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FASA DIAM
• Superose 10 - 800 kD
• Biogel P60 3 - 60 kD
• RIP 30 kD, pilih mana ?
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Ion Exchange Chromatography
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Ion Exchange ChromatographyIn the process of ion exchange, ions that are electrostatically
bound to an insoluble and chemically inert matrix are reversibly replaced by ions in solution.
Anion exchange: R+A- + B- R+B- + A-
Cation exchange: R- A+ + B+ R-B+ + A+
R stands for the resin.
The affinity with which a particular polyelectrolyte binds a given ion exchanger depends on the identities and concentrations of other ions in solution because of the competition among these various ions for the binding sites on the ion exchanger. Because the charge on a polyelectrolyte is highly pH-dependent it follows that the binding affinity of the polyelectrolyte for the resin will be highly pH-dependent. These principles are used to great advantage in isolating biological molecules by ion exchange chromatography.
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Ion exchange resins
1. Cellulose (the cellulose, derived from wood or cotton, is lightly derivatized with ionic groups to form the ion exchanger).
Anion exchange
Cation exchange
2. Gel-type ion exchangers: combine the separation properties of gel filtration with those of ion exchange. Because of their high degree of substitution of charged groups, which results from their porous structures, these gels have a higher loading capacity than do cellulosic ion exchangers.
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Ion Exchange Chromatography
Ion exchange chromatography – binding and separation of proteins based on charge-charge interactions
Proteins bind at low ionic strength, and are eluted at high ionic strength
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++
+
+
++
+ ++
+
-
- -
-
++
+
+
+
++
+
+
+-
- -+
Positively charged(anionic) ion
exchange matrix
Net negatively charged (cationic)
protein at selected pHProtein binds to matrix
Reminder about protein net charge, pI and pH
• All proteins have ionisable groups on the surface (N-terminal amino and carboxylate, Glu, Asp, His, Lys and Arg side chains)
• These groups are charged or neutral depending on pH (e.g., -COO- + H+ COOH)
• The net charge on a protein changes at different pHs
• Each protein has a pH where the net charge is zero (the pI: Isoelectric Point)
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Muatan aa sangat dipengaruhi pH lingkungan
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Useful rules: At pH > pI, protein net charge is negative At pH < pI, protein net charge is positive At pH = pI, protein net charge is zero
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Determining the isoelectric point (pI)
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Isoelectric point: The pI is the pH at which there is zero net charge on a molecule. Look at Asp.
The zero net charge form is a part of the first two ionizations. Therefore, the maximum amount of this is present at a pH of (2.09 + 3.86)/2 = 2.98 = pI.
HOOC-CH2-CH-COOH
NH3+
HOOC-CH2-CH-COO-NH3
+
+ H+ 2.09
HOOC-CH2-CH-COO-NH3
+
-OOC-CH2-CH-COO-NH3
+
-OOC-CH2-CH-COO-NH3
+
-OOC-CH2-CH-COO-NH2
+ H+ 3.86
+ H+ 9.82
Typical ion exchange protein separation
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Loading starts
Loading ends,Low salt wash begins
Protein absorbance
Peak of unbound protein
Salt gradient
0
1M
Salt gradient begins
Salt gradient ends
Eluted peaks of weakly bound (I), moderately bound (II)
and tightly bound (III) proteins
IIIII
I
Ion Exchange (IEX) Chromatography
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PENUKAR ION
Elusi: mengurangi kekuatan ikatan antara protein –fasa diam
• Perubahan pH
• Perubahan kekuatan ion
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Ion Exchange Chromatography (con’d)
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Hydrophobic interaction chromatography
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Prinsip pemisahan:partisi• Koefisien distribusi KD
• Hidrofob-hidrofob
• Hidrofil-hidrofil
• Elusi: gradien/isokratik
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Hydrophobic Interaction Chromatography (HIC)
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Affinity Chromatography
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Affinity chromatography….
• Binding of a protein to a matrix via a protein-specific ligand– Substrate or product analogue
– Antibody– Inhibitor analogue– Cofactor/coenzyme
• Specific protein is eluted by adding reagent which competes with binding
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Affinity Chromatography
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We will use bound Adenosine-5’-monophosphate. This is part Of NAD+. LDH will Bind. Release LDH by adding NADH
Affinity chromatography…..
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Matrix Spacer arm
Affinity ligand
+
Active-site-bound enzyme
1. Substrate analogue affinity chromatography
Matrix Spacer arm
Antibody ligand
+
Antibody-bound enzyme
2. Immunoaffinity chromatography
Protein epitope
Enzyme
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KROMATOGRAFI AFINITAS
AFINITAS BIOSPESIFIK
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AFINITAS LAKTAT DEHIDROGENASE
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IMUNOAFINITAS
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NAD+
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AMP
Affinity chromatography
• Remember: NADH is a co-substrate for lactate dehydrogenase.
• We use AMP-Sepharose: AMP is covalently bound to the affinity gel, which will not pass through the filter.
• LDH binds to the AMP b/c it looks like half an NADH.
• Thus LDH remains immobilized in the column until we offer it something more satisfying.
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Sieving effect
Relative charge
Visualization- staining with dye, fluorescent antibody (Western blotting)
SDS- protein denaturant, enables separation based almost exclusively on molecular weight
Iso-electric focusing- method to measure pI, but also can be used for separation
Gel Electrophoresis
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ISOELECTRICFOCUSING
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Amfolit : oligomer,Poliaminpolikarboksilat
Lar Anoda: asam kuatLar Katoda: basa kuat
Amfolit neg ke anodaMendekati daerah lb asam, ionisasinya menurun, ttp ionisasi ggs amin naik
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Asam amino analisator
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Biakan Sel
Ekstraseluler
Pemisahan sel
Pemekatan
Intraseluler
Pemisahan sel
Pemecahan sel
Pemisahan dinding sel
Pemekatan
K. Penukar ion
K. Interaksi hidrofobik
K. Gel filtrasi
Stabilitas, pengawet, potensi
Sediaan cair/serbuk82
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Protein Concentration
• Lowry ( most cited reference in biology)– Color assay
• A280
– Intrinsic absorbance– Relies on aromatic amino acids
• BCA– Modification of Lowry: increased sensitivity and
consistency
• Bradford– Shifts Amax of dye from 465nm to 595nm
A280
• Uses intrinsic absorbance
• Detects aromatic residues – Resonating bonds
• Depends on protein structure, native state and AA composition
• Retains protein function
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Proteins
Macromolecules built of amino acids.Huge number of possibilitiesClassified in many ways:• solubility• composition• Shape : globular vs fibrous• physical properties• function• 3-D structure
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SolubilityAlbumins Soluble in water and salt
soln’s
Globulins Sparingly soluble in water but soluble in salt solutions
Prolamines Soluble in 70-80% EtOH but insol in water and absolute
EtOH
Histones Soluble in salt soln’s
Scleroproteins Insoluble in water or salt soln’s
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CompositionSimple vs. Conjugated
Simple-Conjugated-
Apoprotein-Holoprotein-Prosthetic group-
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FunctionEnzymatic catalysts
Transport and storage of molecules- Hb, ferritin
Mechanical functions- elastin
Movement- myosin
Protection- Ab
Information processing- rhodopsin
Regulatory- renin
Other90
Structure
Primary (1o)- sequence of amino acids
Secondary (2o)- local 3-D shape
-helix
ß-sheet
collagen triple helix
Tertiary (3o)- global 3-D shape
Quaternary (4o)- relation of polypeptides
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