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Pedoman pemurnian

protein
:
1. Menentukan tujuan

Kemurnian, aktivitas, kuantitas yang diperlukan untuk


produk akhir untuk menghindari kelebihan atau
kekurangan metode yang digunakan.

2. Menentukan sifat (properties) dari protein target

dan batas kritis pengotor, untuk menyederhanakan


teknik seleksi dan optimasi.
3. Menentukan metode analisis yang tepat, untuk
mempercepat deteksi aktivitas/recovery protein agar
pekerjaan efisien.
4. Meminimalkan penanganan sampel pada setiap
tahap

5. Meminimalkan pengunaan zat tambahan


(additives)
6. Menyingkirkan kontaminan yang dapat
meyebabkan kerusakan protein target,misalnya
protease
7. Penggunaan teknik yang berbeda pada masingmasing tahap (berdasar ukuran, muatan,
hidrofobisitas, ligan spesifik)
8. Meminimalkan jumlah tahap purifikasi

Alasan melakukan pemurnian


protein
Untuk menentukan FUNGSI protein
Untuk menentukan STRUKTUR protein
Untuk penggunaan pemurnian pemurnian

product INTERMIDIATE- dalam alur reaksi/


processing
Untuk memproduksi produk COMMERCIAL

STRATEGI PEMURNIAN
PROTEIN
Untuk penggunaan apa protein diproduksi?
Dari organisme apa, pada bagian mana ?
Apakah protein intraseluler atau ekstraseluler?
Jika intraseluler terletak di bagianmana pada sel?
Bagaimana menanganinya?
Berapa kemurnian yang dibutuhkan yang

berhubungan dengan sumber protein dan produk


akhir?
Jika dilakukan Scale-up (skala besar) teknik apa yang
diperlukan, berkaitan efisiensi ; sumber dan
ketersediaan peralatan.

Tiga Tahap Strategi


Purifikasi
Tujuan utama untuk memurniakan protein

adalah untuk mengisolasi, mendapatkan


konsentrasi dan stabiltas protein sesuai dengan
tujuan pemurnian target.
Fase purifikasi intermediate, bertujuan untuk
membuang sebagian pengotor, misalnya protein
lain, asam nukleat, endotoxin, dan virus.
Fase terakhir bertujuan untuk mendapatkan
kemurnian tinggi, menyingkirkan semua pengotor
atau senyawa yang berhubungan dengan protein
target.

Kombinasi Seleksi dan


Tahap
Purifikasi
yang
Untuk menjamin perkembangan
Optimum
metode yang: cepat, waktu yang
singkat untuk mendapatkan
produk yang murni, dan
keuntungan ekonomi.

Preparasi
GOAL (Tujuan akhir) : kemurnian dan kuantitas tinggi,

maintanace (pemeliharaan) aktivitas biologi, sehingga


memenuhi syarat ekonomi dan waktu.
Tentukan kemurnian yang dibutuhkan oleh produk
akhir
Contoh kemurnian yang dibutuhkan :
Kemurnian Sangat Tinggi >99% Penggunaan terapi, in vivo

studies
Kemurnian Tinggi 95-99% , menurut pengujian karakteristik
dengan metode Kristalografi sinar X dan fisiko-kimia
Moderat
< 95%
, pengujian antigen untuk
produksi antibodi pada sekuensing N-terminal

Identifikasi kontaminan kunci/utama


Identifikasi kemungkinan kontaminansi alami yang

tersisa
Lebih baik kemurnian tinggi daripada kurang
Kontaminan yang mendegradasi atau
menginaktifkan protein atau mengganggu dalam
analisis sebaiknya dihilangkan sejak awal
Periksa stabilitas protein, minimal kaitannya
dengan pH dan kekuatan ionik
Seleksi metode pengujian yang cepat dan reliabel.

Seleksi sumber protein


Material dapat berasal dari
Animal tissue
Plant material
Biological fluids (e.g. blood, milk, sera)
Bacteria

RECOMBINANT expression
Fermentation cultures (yeast, fungi, bacteria)
Cell cultures (animal cells, plant cells, insect cells)

PENTING !
Konsentrasi Protein rendah dalam sumber

alami (natural sources), maka


Dibutuhkan induksi ekspresi dalam ekspresinya

Atau dalam teknik rekombinan diekspresikan


dalam berbagai system ekspresi.

Classification of proteins by
structural characterisation
Structural characteristics

Examples

Comments

Monomeric

Lysozyme, growth hormone

Usually extracellular; often have


disulphide bonds

Oligomeric with identical subunits

Glyceralsehyde-3-phosphate
dehydrogenas, catalase, hexokinase

Mostly intracellular enzymes, rarely have


disulphide bonds

Oligomeric with mixed subunits

Petussis toxin

Allosteric enzymes, different subunits


have different functions

Membrane bound peripheral

Mitrochondrial ATPase, alkaline


phosphatise

Readily solubilised in detergents

Membrane bound intergral

Porins, insulin receptors

Requires lipid for stability

Membrane bound conjugated

Glycoproteins, lipoproteins,
nucleoproteins

Many extracellular proteins contain


carbohydrate

Function

Examples

Amino acid storage

Seed proteins (e.g. gluten), milk proteins (e.g. caesin)

Structural inert

Collagen, keratin

Structural with activity

Actin, myosin, tubulin

Binding soluble

Albumin, hemoglobin, hormones

Binding insoluble

Surface receptors (e.g. insulin receptor), antigens (e.g. viral coat proteins)

Binding with activity

Enzymes, membrane transporters (e.g. ion pumps

Relative abundance

Classification of proteins by
function

Protein properties and their effect on


development of purification strategies
Sample and target protein properties

Influence on purification strategy

Temperature stability

Need to work rapidly at lower temperatures

pH stability

Selection of buffers for extraction and purification (conditions for ion exchange, affinity or
reverse phase chromatography)

Organic solvents

Selection of condition for reverse phase chromatography

Detergent requirements

Consider effects on chromatographic steps and the need for detergent removal. consider
choice of detergent

Salt (ionic strength)

Selection of conditions for precipitation techniques and hydrophobic interaction


chromatography

Co-factors for stability or activity

Selection of additives, pH, salts and buffers

Protease sensitivity

Need for fast removal of proteases or addition of inhibitors

Sensitivity to metal ions

Need to add EDTA or EGTA in buffers

Redox sensitivity

Need to add reducing agents

Molecular weight

Selection of gel filtration media

Charge properties

Selection of ion exchange conditions

Biospecific affinity

Selection of ligand for affinity medium

Post translational modifications

Selection of group specific affinity medium

Hydrophobicity

Selection of medium for hydrophobic interaction chromatography

Yields for multi-step protein


purifications
Minimalkan tahap purifikasi
10
0

Optimiasi setiap tahap


Hati-hati terhadap hasil jika
diperlukan beberapa tahap

Tahap kunci dalam


purifikasi
Memisahkan protein target dari material awal
Memisahkan padatan dari protein yang ada

dalam supernatan
Koncentration protein
Membuang/membersihkan kontaminan untuk
meningkatkan kemurnian
Stabilisasi dari target protein

Three phase purification


strategy

Proses final purifikasi yang ideal melakukan preparasi sampel , meliputi


ekstraksi dan klarifikasi yang dibutuhkan, melalui 3 tahap utama
purifikasi.Jumlah tahap purifikasi ditenrukan oleh startegi purifikasi, kemurnian
yang dibutuhkan dan untuk tujuan apa protein digunakan.

Analisis Protein
Pekerjaan yang dilakukan berkaitan dengan

keperluan dan penentuan hasil selama


purifikasi ditentukan oleh
Untuk tujuan apa protein tersebut
Bahan awal sumber diperolehnya protein

tersebut.
Physical studies e.g. x-ray, NMR, EM
End product pharmaceuticals

Analysis kemurnian
protein
Total protein
Specific quantification
Activity assays
Binding assays
Detection of impurities
HPLC
Gel electrophoresis

Protein mass spectrometry

Methods for quantification of


proteins in solution
Assay method

Useful range

NanoOrange assay

100ng/ml to
10ug/ml

BCA method (Cu


reduction)

0.5ug/ml to
1.5mg.ml

Bradford assay (dye


binding)

1ug/ml to 1.5
mg/ml

Lowry assay

1ug/ml to
1.5mg.ml

Absorbance at 280nm

50ng/ml to
2mg/ml

Comments

Samples can be read up to six hours later without any loss in the
sensitivity
Low protein to protein signal variability
Detection not influenced by reducing agents or nucleic acid
Samples must be read within 10 mins
Not compatible with reducing agents
Protein precipitates over time
High protein to protein signal variability
Not compatible with detergents
Lengthy, multi-step procedure
Not compatible with detergents, carbohydrates or reducing agents
High protein to protein signal variability
Detection influenced by nucleic acids and other UV absorbing
contaminants

Protein Purification
Crude extract: breaking cells, by osmosis lysis

or homogenization.
Fractionation: separate proteins into different
fraction based on size of charge.
Salting out: The solubility of proteins is
lowered at high salt concentration.
Ammonium sulfate ((NH4)2SO4).
Dialysis is a procedure to separate proteins
from solvents

TAHAP PURIFIKASI (Ada


Tugas)
[1] Protein Source: 1. Tissue 2. Microbial cells.
Jika kita isolasi protein dari jaringan sel, kita harus
mengisolasi bagian dari sel, misalnya organela
Dapat dilakukan sentrifugasi dengan kecepatan
bertahap/bertingkat
1. Pemecahan sel (Grind tissue (blender/homogenizer)
2. Centrifuge the speed determines which organelles
appear in the pellet and this is based on density
(which ones will pellet first?)

Lyse Cells
Physical
French pressure cell
Sonication
Glass beads

Chemical
Detergents
Enzymes
Hypotonic buffer
http://www.diversified-equipment.com/pics/12130.jpg

[2] Solubilization
1. Osmotic lysis: simple and gentle
- place cells in a hypotonic solution
2. Mechanical: good if cells have cell wall
crushing or grinding of cells,
French press (pressure), sonication (sound)
3. Detergents: used if protein is in lipid membrane
pH: use an appropriate buffer.
Temperature: 0-4 C can be unstable once outside of cell
Proteases: must be inhibited (Proteases (proteinases) cut amide bonds of
protein)
Adsorption: keep concentrated (denatures at surfaces)
minimize foaming/exposure to air (reduce unfolding)
Storage: store under inert gas, frozen at -80 C or -196 C

Stabilize Sample
Control pH
Use appropriate buffer

Control temperature
Keep samples on ice or work in cold room
Prechill instruments

Prevent frothing/foaming
Handle gently.

Maintain concentrated sample

Stabilize Sample
Protease inhibitors
O

Phenylmethylsulfon

yl fluoride (PMSF)
Leupeptin
Aprotinin
Chymostatin
Pepstatin A

S
O
PMSF

Protein Solubility

Salting in

Ions shield charges and

allow proteins to fold.

Ions compete with water

to interact with side


groups. When [salt] is
high enough, salt wins
causing protein to
precipitate.

solubility

Salting out

salting in

salting out

Generally use ammonium

sulfate to precipitate
proteins in the lab.

[salt]

Separate Cell Debris


Centrifugatio

n
Supernate
Pellet

Filter

(Fig 1-8)

[3] Stabilization
Proteins are most stable under pH and ionic
strength close to physiological conditions.
pH: 7.4, (lysosomal enzymes, pH 5)
I (ionic strength) = 0.15 M

[4] Solubilities of Proteins


Salting In
Addition of salt at low ionic strength can increase
solubility of a protein by neutralizing charges on the surface
of the protein, reducing the ordered water around the
protein and increasing entropy of the system.
Salting out (Can be used for Fractionation)
If the concentration of neutral salts is at a high level
(>0.1M), in many instances the protein precipitates.
This phenomenon apparently results because the
excess ions (not bound to the protein) compete with
proteins for the solvent. The decrease in solvation and
neturalization of the repulsive forces allows the proteins to
aggregate and precipitate. This effect is called "salting
out".

The effect of salt on different proteins may differ:


Certain proteins precipitate from solution under
conditions
In which others remain quite soluble.
Once the protein is precipitated (not denatured)
can separate by centrifugation pellet
can be redissolved in buffer for further purification

Salting out
Ammonium sulfate (half saturated)
Ethanol (90%)
salting out adding salt eg ammonium sulfate

salting-out effect
as [salt] less H2O is available for hydration of protein
proteins will aggregate

[5] Dialysis
Following a salting-out step, the solution will
contain a high concentration of salt that can be
disruptive to subsequent chromatographic steps.
The salt can be removed by dialysis dialysis
tubing has pores with a specific molecular
weight cut-off that allows smaller molecules
(salt) to pass.
Buffer large volume
Dialysis tubing with protein and high salt

Exchange buffer
> 3 times

Dialysis

A form of size exclusion

chromatography.
Used to desalt and

concentrate protein
samples.
Dialysis tubing has set

molecular weight cut off.


Only molecules or ions
smaller than MWCO will
move out of the dialysis bag.

Protein molecules (red) are retained within the dialysis bag, whereas
small molecules (blue) diffuse into the surrounding medium.

[6] Separation - Chromatography


Makes use of a mobile phase (fluid - usually buffer)
& a stationary phase (usually small beads).

Ion exchange
chromatography

pH and [salt] dependent


Separates by ionic
charge: cations & anions
Cation exchange
Anion exchange

Where on the pH scale should the buffer


be compared to the pI of the protein?
This is an example of cation exchange.
How will anion exchange work?
Cation exchanger:
CM-cellulose

-CH2-COO-

Anion exchanger:
DEAE-cellulose

C2H5
-CH2-CH2-NH-C2H5
+

Choosing an ion exchanger will depend on:


The pI value of the target protein.
The pH of buffer used.
An (acidic) protein of a pI of 5.
At pH 7 (phosphate buffer or Tris buffer),
the protein will carry a net negative charge.
The protein will bind to an anion exchanger (DEAE)
Will be repelled from a cation exchanger (CM).
Apply the protein mixture containing the target protein.
Remove neutral and basic proteins in flow-through.
Recover the target protein with increasing [salt].

Size exclusion chromatography


Gel filtration chromatography
Contains porous beads
Separates according to size and shape
Larger proteins excluded
from the small pores
Quaternary structure determination,
& Mr estimation using
a standard curve
(log Mr vs elution volume)
Fibrous proteins
Spherical vs rod-shaped proteins

molecular weight, native


molecular
molecularsieve
sievechromatography
chromatography(gel
(gelfiltration,
filtration,molecular
molecular
sizing)
sizing)
light
lightscattering
scattering
hydrodynamic
hydrodynamic(equilibrium
(equilibriumcentrifugation)
centrifugation)

mix

large
molecules
first

Gel Filtration Chromatography. A mixture of proteins in a small volume is


applied to a column filled with porous beads. Because large proteins
cannot enter the internal volume of the beads, they emerge sooner than do
small ones.

Affinity chromatography
Separates by specific interactions
Contains a ligand:
enzyme substrate
receptor hormone
antigen antibody
(His)6 protein Ni2+

Affinity
Chromatography
Mobile phase
Usually liquid

Stationary phase
Receptor bound to

inert bead

Immunoaffinity column

glutathione

GST tag

http://www1.qiagen.com/products/protein/images/fig_LC_flexible_immobilization.gif

Thin Layer
Chromatography
Mobile phase
Nonpolar liquid

Stationary phase
Polar solid material

spread on backing (glass


or thin sheet of metal)

Separates molecules

based on polarity

Thin Layer
Chromatography
Solvent
front

Distance traveled
by solvent

Relative front value


Rf = distance traveled by substance
distance traveled by solvent

High Rf value = nonpolar substance


Low Rf value = polar substance
Origin

Distance traveled
by substance

Reverse Phase
Chromatography
Solvent
front

Mobile phase

Distance traveled
by solvent

Polar liquid

Stationary phase

Distance traveled
by substance

Nonpolar liquid immobilized

on inert solid

High Rf value = polar substance


Low Rf value = nonpolar substance
Origin

High Performance Liquid


Chromatography
Mobile phase
Liquid

Stationary phase
Small diameter particles

packed into column.

Pressure is required to

push liquid through


column.

Advantages
Better resolving power
Faster
http://www.waters.com/watersdivision/ContentD.asp?watersit=JDRS-5LTGBH

High Performance Liquid


Chromatography

http://academic.sun.ac.za/saf/units/aaa/images/image4_l.jpg

Electrophoretic Analysis
Separates according to size and charge concentration
Matrix is polyacrylamide (proteins) or agarose (nucleic acids)
SDS-PAGE (polyacrylamide gel electrophoresis):
uses sodium-dodecyl sulphate (SDS) to coat the proteins
to give them all equivalent (negative) charge concentration
proteins can then separate by M.W. only

Polyacrylamide Gel Electrophoresis.

Polyacrylamide Gel Electrophoresis. The negatively charged SDS (sodium


dodecyl sulfate)-protein complexes migrate in the direction of the anode, at
the bottom of the gel. The sieving action of a porous polyacrylamide gel
separates proteins according to size, with the smallest moving most rapidly.

SDS-PAGE

TrxSTS TrxCHS
sip

sip

97
66

60K
45

31
22
14

ssolublefraction
iinsolublefraction
ppostNi2+column

Estimating the Molecular Weight of a


Protein

Isoelectic focusing
Ampholytes are low M.W. organic acids and bases
that distribute along the electric field across the gel.
Each protein of a mixture distributes across the gel
according to their pI.
Isoelctric focusing can be described as electrophoresis in a pH gradient
set up between a cathode and anode with the cathode at a higher pH than
the anode. Because of the amino acids in proteins, they have amphoteric
propertites and will be positively charged at pH values below their pI and
negatively charged above. This means that proteins will migrate toward
their pI. Most proteins have a pI in the range of 5 to 8.5.
Under the influence of the electrical force the pH gradient will be
established by the carrier ampholytes, and the protein species migrate and
focus (concentrate) at their isoelectric points. The focusing effect of the
electrical force is counteracted by diffusion which is directly proportional to
the protein concentration gradient in the zone. Eventually, a steady state is
established where the electrokinetic transport of protein into the zone is
exactly balanced by the diffusion out of the zone.

Isoelectic focusing

carrier ampholite; oligoamino, ologicarboxylic acids of 600~900 Da


R-NH-(CH2)n -N-(CH2)n-COOH
R=H, -CH2COOH, CH2-N-R2R2

2-D electrophoresis
Perhaps certain proteins have
identical pIs or molecular weights
1. Separate by IEF 1st dimension
2. Separate by SDS-PAGE 2nd dimension
this gives a second dimension to the analysis

Isoelectric Focusing
pI of a protein: net

charge=0
A pH gradient is
established by
allowing a mixture of
organic acids and
bases (ampholytes).
Protein migrates
until it reaches the
pH that matches its
pI

Two-Dimensional
Electrophoresis
Separates

proteins of
identical MW
that differ in pI
or proteins with
similar pI but
different MW.

Monitoring Progress of
Purification Protocol
Total protein (mg)
Quantity of protein present in fraction

Total activity (units of activity)


Use a portion of sample to determine activity.
Multiply activity by total volume to determine

total activity.

Monitoring Progress of
Purification Protocol

Specific activity (units of activity/mg)


Total
S.A.
=

activity
Total protein

% yield: measure of activity retained

after each step in procedure.


% yield =

Total activity at particular step


Total activity of initial extract

Monitoring Progress of
Purification Protocol
Purification level: Measure of increase in

purity of protein throughout procedure.

Purification =

Specific activity at particular step


Specific activity of initial extract

Monitoring Progress of
Purification Protocol
Step

Total protein
(mg)

Total activity
(units)

Specific activity
(units/mg)

Yield (%)

Purification
level

Initial extract

15,000

150,000

10

100

(NH4)2SO4
precipitation

4,600

138,000

30

92

Ionexchange

1,278

115,500

90

77

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)

Characterization of
Protein
Molecular mass
Electrophoresis
Matrix-assisted laser desorption-ionization

time of flight (MALDI-TOF)

Isoelectric point
Isoelectric focusing

3-D Structure
X-ray crystallography

Electrophoresis

Separates

molecules based
on molecular mass
and/or charge.

http://www.science.fau.edu/chemistry/Mari/biochemlab/manual.html

SDS-PAGE
Sodium dodecyl

sulfate polyacrylamide
gel electrophoresis

Separation based on

molecular mass.

Coat samples with

SDS to give uniform


charge to mass ratio.
Makes all proteins

negatively charged.
http://www.activemotif.com/images/products/nited_pchromo2_gel.jpg

MALDI-TOF
Laser displaces

sample into
ionization chamber.
Ions travel through

electrical field.
Heavier ions travel

more slowly.
http://oregonstate.edu/instruction/bb450/stryer/ch04/Slide22.jpg

Isoelectric Focusing

anode

cathode

Separation

based on
charge.
Can be used to

experimentally
determine pI.
http://www.food.rdg.ac.uk/online/fs460/lecture4/l4a.gif

X-ray Crystallography

Electrons in crystal scatter x-rays to produce an image.

Fourier transformation is used to convert raw data into 3-D structure.

Protein DS3 setelah pemanasan 15 menit


(suhu kultur 70C)

= crude protein (cd)

D70 = cd dipanaskan 70 derajat Celsius 200 KDa


150 KDa
D80 = cd dipanaskan80 derajat Celsius
D90 = cd dipanaskan 90 derajat Celsius
D100 = cd dipanaskan 100 derajat
Celsius

120 KDa
100 KDa
85 KDa
70 KDa
60 KDa
50 KDa
40 KDa
30 KDa
25
KDa
20 KDa

D70

D80

D90

D100

Produk Purifikasi Protein dari Isolat DS3 dalam rangka


mendapatkan DNA polimerase termostabil

Gambar Profil hasil pemurnian DNA polimerase dari isolat DS3

pada SDS-PAGE 12% dengan pewarnaan Coomassie Brilliant Blue


R250. M = penanda protein, 1 = protein kasar, 2 = protein kasar
dipanaskan pada suhu 75oc selama 15 menit, 3 = Protein produk
kromatografi penukar ion, 4 = Protein kromatografi gel filtrasi,
= protein yang hilang

Produk amplifikasi DNA dalam PCR oleh protein dari


isolat DS3

Gambar Produk amplifikasi DNA gen caree dalam plasmid PTIS menggunakan
DNA polimerase produk pemurnian. PCR menggunakan primer universal M13 Forward
Amplication Primer, dan primer M13 Reverse Amplication Primer. Elektroforesis
dilakukan pada gel agarose 1% (g/v). M = Penanda DNA BSM13/HinfI, 1 = Mega MixRoyal, 2 = Protein kasar, 3 = Protein kasar dipanaskan 75 oC 15 menit, 4 = Campuran
protein dalam fraksi 8-18 produk kromatografi penukar ion, 5 = protein dalam fraksi
54 produk kromatografi gel filtrasi, 6 = Tanpa protein pengganti Taq polimerase, 7 =
tanpa template DNA.

Konsentrasi BSA
(g/ml)
0
2
4
8
16
32
64

Tahap purifikasi
Protein kasar
Protein yang
dipanaskan suhu 75oC
15 menit
Fraksi 10 hasil
kromatografi penukar
ion
Fraksi 54 hasil
kromatografi gel filtrasi

OD595nm
0
0.085
0.145
0.164
0.383
0.676
1.343

Kerapatan Optik pada


595
0,380
0,315
0,256
0,059

Kadar protein (g/ml)


8,44
6,85
5,41
0,61