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8/18/2019 Purifikasi Protein
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Pedoman pemurnianprotein :1. Menentukan tujuan Kemurnian, aktivitas, kuantitas yang diperlukan untuk
produk akhir untuk menghindari kelebihan ataukekurangan metode yang digunakan.
2. Menentukan sifat (properties) dari protein target
dan batas kritis pengotor, untuk menyederhanakanteknik seleksi dan optimasi.
3. Menentukan metode analisis yang tepat, untukmempercepat deteksi aktivitas/recovery protein agar
pekerjaan efsien.4. Meminimalkan penanganan sampel pada setiap
tahap
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$lasan melakukan pemurnian
protein%ntuk menentukan &%'* protein
%ntuk menentukan +%K+% protein
%ntuk penggunaan pemurnian pemurnianproduct -*'+*0*$+1 dalam alur reaksi/processing
%ntuk memproduksi produk 232*$4
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+$+* P%'*$'
P3+*'• %ntuk penggunaan apa protein diproduksi5
• 0ari organisme apa, pada bagian mana 5
• $pakah protein intraseluler atau ekstraseluler5
• 6ika intraseluler terletak di bagianmana pada sel5
• 7agaimana menanganinya5
• 7erapa kemurnian yang dibutuhkan yangberhubungan dengan sumber protein dan produkakhir5
• 6ika dilakukan cale1up (skala besar) teknik apa yangdiperlukan, berkaitan efsiensi 8 sumber danketersediaan peralatan.
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+iga +ahap trategi
Purifkasi!ujuan utama untuk memurniakan proteinadalah untuk mengisolasi, mendapatkankonsentrasi dan stabiltas protein sesuai dengantujuan pemurnian target.
"ase purikasi intermediate, bertujuan untukmembuang sebagian pengotor, misalnya proteinlain, asam nukleat, endoto9in, dan virus.
"ase terakhir bertujuan untuk mendapatkan
kemurnian tinggi, menyingkirkan semua pengotoratau senyaa yang berhubungan dengan proteintarget.
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Kombinasi eleksi dan +ahap Purifkasi yang3ptimum :%ntuk menjamin perkembanganmetode yang cepat, aktu yang
singkat untuk mendapatkanproduk yang murni, dankeuntungan ekonomi.
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Preparasi#$%& (+ujuan akhir) : kemurnian dan kuantitas tinggi,
maintanace (pemeliharaan) aktivitas biologi, sehinggamemenuhi syarat ekonomi dan aktu.
!entukan kemurnian yang dibutuhkan oleh produkakhir
2ontoh kemurnian yang dibutuhkan :Kemurnian angat +inggi ;
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*dentifkasi kontaminan kunci/utama
*dentifkasi kemungkinan kontaminansi alami yang
tersisa4ebih baik kemurnian tinggi daripada kurang
Kontaminan yang mendegradasi ataumenginakti"kan protein atau mengganggu dalam
analisis sebaiknya dihilangkan sejak aalPeriksa stabilitas protein, minimal kaitannya
dengan pA dan kekuatan ionik
eleksi metode pengujian yang cepat dan reliabel.
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eleksi sumber proteinaterial dapat berasal dari
$nimal tissuePlant material7iological Buids (e.g. blood, milk, sera)7acteria
237*'$'+ e9pression&ermentation cultures (yeast, "ungi, bacteria)2ell cultures (animal cells, plant cells, insect cells)
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P'+*' CKonsentrasi Protein rendah dalam sumber
alami (natural sources), maka
0ibutuhkan induksi ekspresi dalam ekspresinya$tau dalam teknik rekombinan diekspresikandalam berbagai system ekspresi.
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2lassifcation o" proteins bystructural characterisation
Structural characteristics Examples Comments
Monomeric Lysozyme, growth hormone Usually extracellular; often havedisulphide bonds
Oligomeric with identical subunits Glyceralsehyde!phosphatedehydrogenas, catalase, hexo"inase
Mostly intracellular enzymes, rarely havedisulphide bonds
Oligomeric with mixed subunits #etussis toxin $llosteric enzymes, different subunitshave different functions
Membrane bound % peripheral Mitrochondrial $ase, al"aline phosphatise
'eadily solubilised in detergents
Membrane bound % intergral #orins, insulin receptors 'e(uires lipid for stability
Membrane bound % con)ugated Glycoproteins, lipoproteins,nucleoproteins
Many extracellular proteins containcarbohydrate
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2lassifcation o" proteins by"unction
Function Examples
$mino acid storage *eed proteins +eg gluten-, mil" proteins +eg caesin-
*tructural % inert .ollagen, "eratin
*tructural % with activity $ctin, myosin, tubulin
/inding % soluble $lbumin, hemoglobin, hormones
/inding % insoluble *urface receptors +eg insulin receptor-, antigens +eg viral coat proteins-
/inding % with activity 0nzymes, membrane transporters +eg ion pumps
, e l a t i v e
a b
u n d a n c e
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Protein properties and their eDect ondevelopment o" purifcation strategies
Sample and target protein properties Influence on purification strategy
&emperature stability 1eed to wor" rapidly at lower temperatures
p2 stability *election of buffers for extraction and purification +conditions for ion exchange, affinity orreverse phase chromatography-
Organic solvents *election of condition for reverse phase chromatography
3etergent re(uirements .onsider effects on chromatographic steps and the need for detergent removal considerchoice of detergent
*alt +ionic strength- *election of conditions for precipitation techni(ues and hydrophobic interactionchromatography
.ofactors for stability or activity *election of additives, p2, salts and buffers
#rotease sensitivity 1eed for fast removal of proteases or addition of inhibitors
*ensitivity to metal ions 1eed to add 03&$ or 0G&$ in buffers
'edox sensitivity 1eed to add reducing agents
Molecular weight *election of gel filtration media
.harge properties *election of ion exchange conditions
/iospecific affinity *election of ligand for affinity medium
#ost translational modifications *election of group specific affinity medium
2ydrophobicity *election of medium for hydrophobic interaction chromatography
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Eields "or multi1step proteinpurifcations
FGG
4 Minimal"an tahap purifi"asi
4 Optimiasi setiap tahap
4 2atihati terhadap hasil )i"adiperlu"an beberapa tahap
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+ahap kunci dalampurifkasiemisahkan protein target dari material aal
emisahkan padatan dari protein yang ada
dalam supernatanKoncentration protein
embuang/membersihkan kontaminan untukmeningkatkan kemurnian
tabilisasi dari target protein
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+hree phase purifcationstrategy
#roses final purifi"asi yang ideal mela"u"an preparasi sampel , meliputie"stra"si dan "larifi"asi yang dibutuh"an, melalui ! tahap utama purifi"asi5umlah tahap purifi"asi ditenru"an oleh startegi purifi"asi, "emurnianyang dibutuh"an dan untu" tu)uan apa protein diguna"an
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$nalisis ProteinPekerjaan yang dilakukan berkaitan dengan
keperluan dan penentuan hasil selamapurifkasi ditentukan oleh%ntuk tujuan apa protein tersebut
7ahan aal sumber diperolehnya proteintersebut. Physical studies e.g. 91ray, ',
nd product - pharmaceuticals
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$nalysis kemurnianprotein +otal protein
pecifc Huantifcation
$ctivity assays7inding assays
0etection o" impuritiesAP42
el electrophoresis
Protein mass spectrometry
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$ssay method Useful range .omments
1anoOrange assay 677ng8ml to67ug8ml •*amples can be read up to six hours later without any loss in the
sensitivity•Low protein to protein signal variability•3etection not influenced by reducing agents or nucleic acid
/.$ method +.ureduction-
79ug8ml to69mgml •*amples must be read within 67 mins
• 1ot compatible with reducing agents
/radford assay +dye binding-
6ug8ml to 69mg8ml •#rotein precipitates over time
•2igh protein to protein signal variability• 1ot compatible with detergents
Lowry assay 6ug8ml to69mgml •Lengthy, multistep procedure
• 1ot compatible with detergents, carbohydrates or reducing agents
$bsorbance at :7nm 97ng8ml to:mg8ml •2igh protein to protein signal variability
•3etection influenced by nucleic acids and other U< absorbingcontaminants
ethods "or Huantifcation o"proteins in solution
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Protein Purifcation
2rude e9tract: breaking cells, by osmosis lysisor homogeniIation.&ractionation: separate proteins into diDerent
"raction based on siIe o" charge.
alting out: +he solubility o" proteins isloered at high salt concentration.$mmonium sul"ate (('AJ)K3J).
0ialysis is a procedure to separate proteins"rom solvents
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+$A$P P%*&*K$* ($da +ugas)[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
bertahapbertingkat
1. !emecahan sel "#rind tissue "blenderhomogeni$er%
2. &entrifuge ' the speed determines (hich organellesappear in the pellet and this is based on density"(hich ones (ill pellet first)%
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4yse 2ellsPhysical&rench pressure cell
onicationlass beads
2hemical0etergents
nIymes
Aypotonic buDerhttp://.diversifed1eHuipment.com/pics/FFLG.jpg
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tabiliIe ample
2ontrol pA%se appropriate buDer
2ontrol temperatureKeep samples on ice or ork in cold roomPrechill instruments
Prevent "rothing/"oamingAandle gently.
aintain concentrated sample
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tabiliIe ampleProtease inhibitors
Phenylmethylsul"onyl Buoride (P&)
4eupeptin
$protinin
2hymostatin
Pepstatin $
S
O
O
F
!M6
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Protein olubilityalting in *ons shield charges and
allo proteins to "old.
alting out *ons compete ith ater
to interact ith sidegroups. Mhen NsaltO ishigh enough, salt inscausing protein to
precipitate.
enerally use ammoniumsul"ate to precipitateproteins in the lab.
:salt;
s o l u b i l i t y
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eparate 2ell 0ebris
2entri"ugationupernate
Pellet
&ilter
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"ig 17%
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[3] Stabilization
!roteins are most stable under p0 and ionic
strength close to physiological conditions.p0+ >., "lysosomal en$ymes, p0 ?%
I "ionic strength% @ .1? M
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[4] Solubilities of Proteins
Salting In
4ddition of salt at lo( ionic strength can increase
solubility of a protein by neutrali$ing charges on the surface
of the protein, reducing the ordered (ater around the
protein and increasing entropy of the system.
Salting out "&an be used for ractionation)
Af the concentration of neutral salts is at a high le-el
"B.1M%, in many instances the protein precipitates.
This phenomenon apparently results because thee5cess ions "not bound to the protein% compete (ith
proteins for the sol-ent. The decrease in sol-ation and
neturali$ation of the repulsi-e forces allo(s the proteins to
aggregate and precipitate. This effect is called Csalting
outC.
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Salting outAmmonium sulfate (half saturated)
thanol (!"#)•
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[$] %ial&sis
• ollo(ing a saltingout step, the solution (ill
contain a high concentration of salt that can be
disrupti-e to subseuent chromatographic steps.• The salt can be remo-ed by dialysis ' dialysis
tubing has pores (ith a specific molecular
(eight cutoff that allo(s smaller molecules
"salt% to pass.
E5change buffer
B / times
Fuffer' large -olume
Dialysis tubing (ith protein and high salt
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0ialysis$ "orm o" siIe e9clusionchromatography.%sed to desalt and
concentrate protein
samples.
0ialysis tubing has setmolecular eight cut oD.3nly molecules or ions
smaller than M23 illmove out o" the dialysis bag.
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!rotein molecules "red% are retained (ithin the dialysis bag, (hereas
small molecules "blue% diffuse into the surrounding medium.
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['] Searation *hromatograh&
Makes use of a mobile phase "fluid usually buffer%
G a stationary phase "usually small beads%.
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p0 and :salt; dependent• 6eparates by ionic
charge+ cations G anions
&ation e5change
4nion e5change
Aon e5change
chromatography
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Hhere on the p0 scale should the buffer
be compared to the pA of the protein)
This is an e5ample of cation e5change.
0o( (ill anion e5change (ork)
&ation e5changer+&Mcellulose &02&**
4nion e5changer+
&20
?
DE4Ecellulose &02&0
2I0&
20
?
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&hoosing an ion e5changer (ill depend on+
The pA -alue of the target protein.
The p0 of buffer used.
4n "acidic% protein of a pA of ?.
4t p0 > "phosphate buffer or Tris buffer%,the protein (ill carry a net negati-e charge.⇒ The protein (ill bind to an anion e5changer "DE4E%⇒ Hill be repelled from a cation e5changer "&M%.
• 4pply the protein mi5ture containing the target protein.• Kemo-e neutral and basic proteins in flo(through.• Keco-er the target protein (ith increasing :salt;.
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Size e+clusion chromatograh&
,el filtration chromatograh&
• &ontains porous beads
• 6eparates according to si$e and shape• Larger proteins e5cluded
from the small pores
• uaternary structure determination, G Mr estimation using
a standard cur-e
"log Mr vs elution -olume%
Ⓠ ibrous proteins6pherical vs rodshaped proteins
l l i ht ti
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molecular (eight, nati-emolecular sie-e chromatograh& (gel filtration. molecular
sizing)
light scattering
h&drod&namic (e/uilibrium centrifugation)
molecular sie-e chromatograh& (gel filtration. molecularsizing)
light scattering
h&drod&namic (e/uilibrium centrifugation)
mi+
largemoleculesfirst
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,el 0iltration *hromatograh& 4 mi5ture of proteins in a small -olume is
applied to a column filled (ith porous beads. Fecause large proteins
cannot enter the internal -olume of the beads, they emerge sooner than do
small ones.
4ffi it h t h
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• 6eparates by specific interactions
• &ontains a ligand+
en$yme substratereceptor hormoneantigen antibody"0is%
9 protein ' Ii2
4ffinity chromatography
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$Qnity
2hromatographyobile phase
%sually liHuid
tationary phase
eceptor bound toinert bead
Ammunoaffinity column
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http://F.Hiagen.com/products/protein/images/fgR42RBe9ibleRimmobiliIation.gi"
glutathione
,S tag
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+hin 4ayer
2hromatography
elative "ront value
" S distance traveled by substancedistance traveled by solvent
Aigh " value S nonpolar substance4o " value S polar substance
*rigin
6ol-ent
front
Distance tra-eled
by substance
Distance tra-eled
by sol-ent
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everse Phase
2hromatography
obile phase
Polar liHuid
tationary phase'onpolar liHuid immobiliIed
on inert solid
Aigh " value S polar substance
4o " value S nonpolar substance
*rigin
6ol-ent
front
Distance tra-eled
by substance
Distance tra-eled
by sol-ent
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Aigh Per"ormance 4iHuid
2hromatographyobile phase
4iHuid
tationary phasemall diameter particles
packed into column.
Pressure is reHuired topush liHuid throughcolumn.
$dvantages7etter resolving poer&aster
http://.aters.com/atersdivision/2ontent0.asp5atersitS601>4+7A
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Aigh Per"ormance 4iHuid
2hromatography
http://academic.sun.ac.Ia/sa"/units/aaa/images/imageJRl.jpg
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Electrophoretic 4nalysis6eparates according to si$e and charge concentration
Matri5 is polyacrylamide "proteins% or agarose "nucleic acids%
6D6!4#E "polyacrylamide gel electrophoresis%+
uses sodiumdodecyl sulphate "6D6% to coat the proteins
to gi-e them all eui-alent "negati-e% charge concentration
' proteins can then separate by M.H. only
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Pol&acr&lamide ,el lectrohoresis
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Pol&acr&lamide ,el lectrohoresis The negati-ely charged 6D6 "sodium
dodecyl sulfate%protein comple5es migrate in the direction of the anode, at
the bottom of the gel. The sie-ing action of a porous polyacrylamide gel
separates proteins according to si$e, (ith the smallest mo-ing most rapidly.
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ti ti th l l M i ht "
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stimating the olecular Meight o" aProtein
Asoelectic focusing
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g
• 4mpholytes are lo( M.H. organic acids and bases
that distribute along the electric field across the gel.
• Each protein of a mi5ture distributes across the gelaccording to their pA.
soelctric focusing can be described as electrophoresis in a p0 gradient
set up bet(een a cathode and anode (ith the cathode at a higher p0 than
the anode. Fecause of the amino acids in proteins, they ha-e amphotericpropertites and (ill be positi-ely charged at p0 -alues belo( their pA and
negati-ely charged abo-e. This means that proteins (ill migrate to(ard
their pA. Most proteins ha-e a pA in the range of ? to 7.?.
Nnder the influence of the electrical force the p0 gradient (ill be
established by the carrier ampholytes, and the protein species migrate and
focus "concentrate% at their isoelectric points. The focusing effect of theelectrical force is counteracted by diffusion (hich is directly proportional to
the protein concentration gradient in the $one. E-entually, a steady state is
established (here the electrokinetic transport of protein into the $one is
e5actly balanced by the diffusion out of the $one.
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2D electrophoresis
!erhaps certain proteins ha-e
identical pAs or molecular (eights
1. 6eparate by AE ' 1st dimension
2. 6eparate by 6D6!4#E ' 2nd dimension
this gi-es a second dimension to the analysis
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*soelectric &ocusingp* o" a protein: net
chargeSG
$ pA gradient isestablished byalloing a mi9ture o"organic acids andbases (ampholytes).Protein migratesuntil it reaches thepA that matches itsp*
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onitoring Progress o"
Purifcation Protocol!otal protein 'mg(
Tuantity o" protein present in "raction
!otal a)ti*ity 'units of a)ti*ity(
%se a portion o" sample to determine activity.
ultiply activity by total volume to determinetotal activity.
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onitoring Progress o"
Purifcation Protocol+pe)i) a)ti*ity 'units of a)ti*ity,mg(
+otal activity
+otal protein
yield measure o" activity retaineda"ter each step in procedure.
6.4. @
Q yield @Total acti-ity at particular step
Total acti-ity of initial e5tract
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onitoring Progress o"
Purifcation Protocol
Puri)ation le*el easure o" increase in
purity o" protein throughout procedure.
!urification @
6pecific acti-ity at particular step
6pecific acti-ity of initial e5tract
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01P$odium dodecyl
sul"ate polyacrylamidegel electrophoresis
eparation based onmolecular mass.
2oat samples ith
0 to give uni"ormcharge to mass ratio.akes all proteins
negatively charged.
http://.activemoti".com/images/products/nitedRpchromoRgel.jpg
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$40*1+3&4aser displaces
sample into
ioniIation chamber.
*ons travel throughelectrical feld.
Aeavier ions travelmore sloly.
http://oregonstate.edu/instruction/bbJ>G/stryer/chGJ/lide.jpg
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*soelectric &ocusing
eparationbased oncharge.
2an be used toe9perimentallydetermine p*.
http://."ood.rdg.ac.uk/online/"sJG/lectureJ/lJa.gi"
anode cathode
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Sray &rystallography Electrons in crystal scatter 5rays to produce an image.
ourier transformation is used to con-ert ra( data into /D structure.
Protein /+3 setelah pemanasan 15 menit
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200 KDa
150 KDa
120 KDa
100 KDa 5 KDa
!0 KDa
"0 KDa
50 KDa
#0 KDa
$0 KDa
25
KDa 20 KDa
7 % %8" %9" %!" %1""
'suhu kultur 0(
D @ crude protein "cd%
D> @ cd dipanaskan > derajat &elsius
D7 @ cd dipanaskan7 derajat &elsius
D8 @ cd dipanaskan 8 derajat &elsius
D1 @ cd dipanaskan 1 derajat
&elsius
#rodu" #urifi"asi #rotein dari >solat 3*! dalam rang"ad t" 31$ li t t bil
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#ambar Profl hasil pemurnian 0'$ polimerase dari isolat 0Lpada 01P$ F= dengan pearnaan 2oomassie Brilliant BlueR250. S penanda protein, F S protein kasar, S protein kasardipanaskan pada suhu !>oc selama F> menit, L S Protein produkkromatograf penukar ion, J S Protein kromatograf gel fltrasi,
S protein yang hilang
mendapat"an 31$ polimerase termostabil
%rodu& amplifi&asi D'( dalam %C) oleh protein dari
i l t DS$
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#ambar Produk amplifkasi 0'$ gen caree dalam plasmid P+* menggunakan0'$ polimerase produk pemurnian. P2 menggunakan primer universal FL Forward
Amplication Primer , dan primer FL Reverse Amplication Primer . lektro"oresisdilakukan pada gel agarose F= (g/v). S Penanda 0'$ 7FL/Hinf *, F S ega i91oyal, S Protein kasar, L S Protein kasar dipanaskan !>o2 F> menit, J S 2ampuranprotein dalam "raksi #1F# produk kromatograf penukar ion, > S protein dalam "raksi>J produk kromatograf gel fltrasi, S +anpa protein pengganti Taq polimerase, ! S
tanpa template 0'$.
isolat DS$
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Konsentrasi *S(/D
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+,g-ml./D55nm
7 7: 779? 76?9
76@?6@ 7!!!: 7@A@@? 6!?!
&ahap purifi"asi Berapatan Opti" padaC9D9 Badar protein +Eg8ml-
#rotein "asar #rotein yangdipanas"an suhu A9o.69 menit
Fra"si 67 hasil"romatografi penu"arionFra"si 9? hasil"romatografi gel filtrasi
7,!7
7,!69
7,:9@
7,79D
,??
@,9
9,?6
7,@6