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BIOKIMIA

III. PROTEIN PURIFICATION


Laboratorium Kimia Medisinal Bagian Kimia Farmasi Fakultas Farmasi UGM

Cakupan
Sifat protein Metode pemisahan protein:
Fraksinasi Pengendapan Kromatografi

Penentuan bobot molekul protein.

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 that work together as a functional unit.
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Protein Concentration from Absorbance


Beers Law A = cl The protein absorbance measured at 280 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 FRAKSINASI
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PEMURNIA N

How to Separate These Objects

Shape Size wood stone Density

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

2 3
6
7 8
Density

Shape
cotton

4
8

4 5 6
9
10 11

wood

7
12

stone

Sieving different sizes

Different sedimentation

Different rolling speed


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Juang RH (2004) BCbasics

Basic Principles of Protein Purification

Cell
Small 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 7
Juang RH (2004) BCbasics

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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Animal feed enzyme


Fermentation
Centrifugation to remove cells

Analytical enzyme
Fermentation

Therapeutic protein
Fermentation

Centrifugation to remove cells

Centrifugation to remove medium

Culture supernatant

Culture supernatant
Protein precipitation

Cell pellet
Cell lysis Centrifugation

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


steps

Intracellular fraction
Protein precipitation

Semi-purified protein
Lyophilisation Bottling

Protein fraction
3-4 purification steps

To chemicals market

Homogeneous protein
Sterile 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 drying
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Solubility of Proteins
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)14

Solubility of Proteins
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, 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.
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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.
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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
Separation of very large from very small molecules is based on an attempt to equilibrate concentration. Osmotic pressure
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Chromatography
1. 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 later
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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 Chromatography


In 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.
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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
+ + + + + + + + + + -+ + + + + + + + + + -+

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

Protein binds to matrix


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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)


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.
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Typical ion exchange protein separation


Loading ends, Low salt wash begins 1M Salt gradient

Protein absorbance

II
Loading starts Peak of unbound protein Salt gradient begins

III

Salt gradient ends

I
Eluted peaks of weakly bound (I), moderately bound (II) and tightly bound (III) proteins
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Ion Exchange (IEX) Chromatography

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PENUKAR ION

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Elusi: mengurangi kekuatan ikatan antara


protein 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 binding


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Affinity Chromatography
We 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 chromatography


Affinity ligand Enzyme

+
Matrix Spacer arm

Active-site-bound enzyme

2. Immunoaffinity chromatography
Antibody ligand Protein epitope

+
Matrix Spacer arm

Antibody-bound enzyme
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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+

AMP
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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 Electrophoresis
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

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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/serbuk
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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 595nm
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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 possibilities Classified in many ways:
solubility composition Shape : globular vs fibrous physical properties function 3-D structure
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Solubility
Albumins 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. Conjugated


SimpleConjugatedApoproteinHoloproteinProsthetic group-

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Function
Enzymatic catalysts Transport and storage of molecules- Hb, ferritin Mechanical functions- elastin Movement- myosin Protection- Ab Information processing- rhodopsin Regulatory- renin Other
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Structure
Primary (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 polypeptides
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