3 Teknik Pemurnian Protein Muthi

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Transcript of 3 Teknik Pemurnian Protein Muthi

BIOKIMIA

III. PROTEIN PURIFICATIONLaboratorium Kimia Medisinal Bagian Kimia Farmasi Fakultas Farmasi UGM

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Cakupan Sifat protein Metode pemisahan protein:Fraksinasi Pengendapan Kromatografi

Penentuan bobot molekul protein.

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Methods in Protein ChemistryThese 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 functionalinformation about a group of proteins that work together as a functional unit.3

Protein Concentration from AbsorbanceBeers Law A = cl The protein absorbance measured at 280 nm is due to Tyr & Trp4

Protein Purification Starting MaterialStart 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 FRAKSINASI5

PEMURNIA N

How to Separate These Objects

Shape Size wood stone Density

1

2 3 4 5 6

7 8 9

10 11

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cotton wood wood cotton stone wood stone cotton stone co

Size

1

2 367 8Density

Shapecotton

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4 5 6910 11

4

5

8

wood

712

stone

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Sieving different sizes

Different sedimentation

Different rolling speed6Juang RH (2004) BCbasics

Basic Principles of Protein Purification

CellSmall molecule

Homogenization

Organelle Cell Debris

Macromolecule(Lipid)

Amino acid, Nucleic acid ProteinCarbohydrate Sugar, Nucleotides, etc Ammonium sulfate fractionation

Size

Charge

Polarity

Affinity

Ion exchange, Reverse phase Gel filtration, chromatography, Affinity Chromatofocusing, chromatography, SDS-PAGE, HIC, Disc-PAGE, Hydroxyapatite Ultrafiltration Salting-out Isoelectric focusing 7Juang RH (2004) BCbasics

Begin with intact tissue Disrupt Blender, homoginizer

Remove debris Centrifugation

Precipitate/concentrate Ammonium sulfate

Purify Chromatography

Analyze Activity, molecular weight8

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 scale9

Downstream process depends on product use1. 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 purified10

Animal feed enzymeFermentationCentrifugation to remove cells

Analytical enzymeFermentation

Therapeutic proteinFermentation

Centrifugation to remove cells

Centrifugation to remove medium

Culture supernatant

Culture supernatantProtein precipitation

Cell pelletCell lysis Centrifugation

Liquid preparation Protein fraction to animal feed 1 or 2 purification marketsteps

Intracellular fractionProtein precipitation

Semi-purified proteinLyophilisation Bottling

Protein fraction3-4 purification steps

To chemicals market

Homogeneous proteinSterile bottling

To pharmaceuticals market

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Centrifugation

Separation of a cell homogenate.

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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 drying13

Solubility of ProteinsSalting 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)14

Solubility of ProteinsSalting 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, 15 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.16

Salting in

Salting out

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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.18

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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DialysisSeparation of very large from very small molecules is based on an attempt to equilibrate concentration. Osmotic pressure21

Chromatography1. Size- sieve effect, small molecules faster 2. Ion exchange- charge attraction at protein surface Choose + stationary phase for proteins with more - charge First bind everything, then elute with salt 1. Hidrophobicity- Reversed Phase HPLC 2. Affinity chromatography Antibody, binding protein Inserted tag (e.g. 6-His)

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Gel Filtration Chromatography

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Gel Filtration size separation

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Principles of gel chromatography (cond)

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Gel Filtration Elution Volumes as a Function of Molecular Weight

Adapted from T. E. Creighton, Proteins, W.H.Freeman,1984.

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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

Large protein

Small protein

GPC in operation

Short path length

Longer path length

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Gel Filtration (GF) Chromatography

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FASA DIAM Superose Biogel P60 10 - 800 kD 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: Cation exchange: R +A - + B - R +B - + A 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.37

Ion exchange resins1. 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.38

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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+ + + + + + + + + + -+ + + + + + + + + + -+

Positively charged Net negatively (anionic) ion charged (cationic) exchange matrix protein at selected pH

Protein binds to matrix40

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)41

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 zero43

Determining the isoelectric point (pI)Isoelectric point: The pI is the pH at which there is zero net charge on a molecule. Look at Asp.NH3+ HOOC-CH2-CH-COOH NH3+-

NH3+ HOOC-CH2-CH-COO NH3+-

+ H+

2.09

HOOC-CH2-CH-COO-

OOC-CH2-CH-COO NH2

+ H+

3.86

NH3+

OOC-CH2-CH-COO

-

-

OOC-CH2-CH-COO

-

+ H+

9.82

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.44

Typical ion exchange protein separationLoading ends, Low salt wash begins 1M Salt gradient

Protein absorbance

0

IILoading starts Peak of unbound protein Salt gradient begins

III

Salt gradient ends

IEluted peaks of weakly bound (I), moderately bound (II) and tightly bound (III) proteins45

Ion Exchange (IEX) Chromatography

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PENUKAR ION

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Elusi: mengurangi kekuatan ikatan antaraprotein fasa diam

Perubahan pH Perubahan kekuatan ion

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Ion Exchange Chromatography (cond)

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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 binding57

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Affinity ChromatographyWe will use bound Adenosine -5-monophosphate. This is part Of NAD+. LDH will Bind. Release LDH by adding NADH

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Affinity chromatography..

1. Substrate analogue affinity chromatographyAffinity ligand Enzyme

+Matrix Spacer arm

Active-site-bound enzyme

2. Immunoaffinity chromatographyAntibody ligand Protein epitope

+Matrix Spacer arm

Antibody-bound enzyme60

KROMATOGRAFI AFINITAS

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AFINITAS BIOSPESIFIK

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AFINITAS LAKTAT DEHIDROGENASE

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IMUNOAFINITAS

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NAD+

AMP65

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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Gel ElectrophoresisSieving 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

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ISOELECTRICFOCUSING

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Amfolit : oligomer, Poliaminpolikarboksil at Lar Anoda: asam kuat Lar Katoda: basa kuat

Amfolit neg ke anoda Mendekati daerah lb asam, ionisasinya menurun, ttp ionisasi ggs amin naik

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Asam amino analisator

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Biakan Sel Intraseluler Pemisahan sel Pemecahan sel Pemisahan dinding sel Pemekatan K. Penukar ion Pemekatan K. Interaksi hidrofobik K. Gel filtrasi Stabilitas, pengawet, potensi Sediaan cair/serbuk82

Ekstraselule r

Pemisahan sel

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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 595nm85

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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ProteinsMacromolecules built of amino acids. Huge number of possibilities Classified in many ways: solubility composition Shape : globular vs fibrous physical properties function 3-D structure87

SolubilityAlbumins solns Globulins but Prolamines Soluble in water and salt Sparingly soluble in water soluble in salt solutions Soluble in 70-80% EtOH but insol in water and absolute

EtOH Histones Soluble in salt solns Scleroproteins Insoluble in water or salt solns

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Composition Simple vs. ConjugatedSimpleConjugatedApoproteinHoloproteinProsthetic 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

StructurePrimary (1o)- sequence of amino acids Secondary (2o)- local 3-D shape -helix -sheet collagen triple helix Tertiary (3o)- global 3-D shape Quaternary (4o)- relation of polypeptides91