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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