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PROTEIN TECHNOLOGY

Microorganism

Fungi

Insect

Plants

Protein

animal
Mammalian

Microorganism
Advantages :
The fermentation process are well understood
Can be cultured in large quantities in a very short time
Can be genetically altered easily in form a fusion
protein break the target protein away from the rest
of fusion protein
Intracellular protein E. coli, extracellular proteins
B. subtilis
Disadvantages :
Unable to carry out certain process, like glycosylation

Fungi
Advantages :
Fungi are capable of some posttranslational modification,like
glycosylation
Disadvantages :
Glycosylation of proteins is slightly
different than in animal cells

Plants
Advantages :
Plants can be genetically modified to make them
produce spesific desired proteins tobacco
Disadvantages :
Not all proteins can be expressed in plants
Plants have tough cell walls extracting
proteins can be time consuming and difficult
Glycosylation of proteins is slightly different
than in animal cells

Mammalian cell systems


Advantages :
Still the best
Disadvantages :
More complex, grow relative slowly, larger
number cells required to seed bioreactors
contamined

Whole animal production systems

Rat monoclonal antibody production


Transgenic animals milk or eggs

Insect system

Baculoviruses ared used as vehicles to


insert DNA to insect cell cultures or
insect larvae
Glycosylation of proteins is slightly
different than in mammal cells.

Protein Purity
Purity : FDA requires that a
sample be composed 99.99%
of target protein.
Sel mengandung ribuan protein
Prosedur pemurnian harus membedakan
protein target dari protein lainnya
Sifat protein merupakan kunci dalam
menentukan proses pemurnian

SISTEM REGULASI PROKARIOT


INDUKSI
NON INDUKSI

SENYAWA PENGINDUKSI

Contoh Sistem Regulasi Overproduksi


Senyawa penginduksi :
IPTG (analog laktosa)

Repressor protein model


Operon:
operator, promoter & genes they
control

RNA
polymerase

serve as a model for gene


regulation
RNA repressor
TATA
polymerase

promoter

operator

gene1

gene2

gene3

gene4

DNA

Repressor protein turns off gene by


blocking RNA polymerase binding site.

repressor

repressor protein

Inducible operon: lactose


Digestive pathway model

RNA
polymerase

When lactose is present, binds to


lac repressor protein & triggers
repressor to release DNA

RNA repressor
TATA
polymerase

gene1

induces transcription
gene2
gene3
gene4

repressor

promoter

operator

DNA

repressor protein
lactose

repressor

lactose repressor protein


complex
conformational change in
repressor protein!

OVERPRODUKSI
STARTER

1%
370C, 150 rpm
OD600nm = 0,6-0,7
NI

370C,150rpm
, on

NI

Panen sel
7000rpm,10,40C
pelet
SDS
PAGE

10000rpm, 15
Ekstrak
protein

pelet
Hasil sonikasi

TAHAPAN UMUM ISOLASI PROTEIN


SUMBER PROTEIN (MIKROBA, TANAMAN, HEWAN)
PROTEIN INTRASELULER

PROTEIN EKSTRASELULER

PEMECAHAN SEL

PEMISAHAN SEL

PEMISAHAN SERPIHAN SEL

PEMEKATAN PROTEIN

PEMEKATAN PROTEIN

PEMURNIAN PROTEIN

LOKASI PROTEIN
PROTEIN INTRASELULER :
Sitoplasma
Periplasma
PROTEIN EKSTRASELULER :
medium

ISOLASI PROTEIN EKSTRASEL

KET :
PROTEIN
SEL

SENTRIFUGA

SUPERNATAN DIAMBIL

ISOLASI PROTEIN INTRASEL

SENTRIFUGA
(pelet sel diambil)

Sel disuspensi
Volume
kecil

SUPERNATAN
sonikasi

sentrifuga

(PROTEIN)

Protein Purifications
Methods
Protein purifications methods :
Preparing the extract for purification
Stabilizing the proteins in solution
Separating the component in the
extract

Preparing the extract for purifications


Cell lysis or disrupting the cell to
release the intracelullar protein
Methods :
Freezing and thawing the cells
Using detergents
Mechanical options :ultrasonics,
homogenizer, grinding with tiny
glass beads, French pressure cell

Cell lysis

HOMOGENIZER

SONICATOR
GLASS BEAD
HOMOGENIZER

FRENCH PRESSURE CELL

PROTEASE INHIBITORS : inaktivasi


protease yang dibebaskan selama lisis sel
PMSF (Phenylmethylsulfonyl fluoride) : inhibits serin
(chymotrypsin, trypsin and thrombin) and thiol proteases (papain)
EDTA (ethylenediamine tetraacetic acid) : inhibits
metalloprotease
EDTA (ethylenediamine tetraacetic acid) : inhibits
metalloprotease
Pepstatin A: inhibits acid protease such as pepsin, renin,
cathepsin D, and chymosin.
Leupeptin : inhibits serin and thiol protease
Aprotinin : inhibits serin protease

Separating the component in the extract

Protein precipitation
Filtration (sized based) separations
methods
Chromatography
Electrophoresis
Analytical methods

Protein precipitation

Salts, most commonly ammonium


sulphate, can be added to the protein
mixture to precipitate the proteins
separates the proteins away from non
proteins

Ammonium sulphate react to stainless


steels ethanol, isopropanol, diethyl
ether and acetone

Strategi pemurnian
Muatan : kromatografi penukar ion
Kepolaran : kromatografi in teraksi hidrofobik
Ukuran : dialisis, ultrafiltrasi, gel elektroforesis,
kromatografi filtrasi gel, dan ultrasentrifugasi
Spesifitas : kromatografi afinitas

Filtration (size-based) separations


methods
Separate molecules on the basis and
density.
Methods :
Centrifugation
Membrane filtration
Dialysis
Diafiltration

Membrane filtration

Thin membranes of nylon or other


engineered substances with very small
pore structures are used to filter out all of
the celullar debris from solution.
Microfiltration : removes the precipitate
and bacteria
Ultrafiltration : filters can catch molecules
such as proteins and nucleic acids.
Limitation : tendency to clog easily.

Dialysis

Passage of solutes through a semipermeable membrane from areas of


higher concentration to the lowest
concentration
Pores in the dialysis membrane are of a
certain size.
This step is required to remove the salts,
solvents and other additive used earlier in
the process.

Diafiltration

Diafiltration adds a filtering component


to dialysis.

Column chromatography
Proteinmixtureappliedtocolumn
Solvent(buffer)appliedtotop,flowed
throughcolumn
Differentproteinsinteractwithmatrixto
differentextents,flowatdifferentrates
Proteinscollectedseparatelyindifferent
fractions

Gel filtration chromatography


- separation by size
Beads have different size pores
As column flows:

large proteins excluded from


pores
and therefore flow rapidly

small proteins enter pores and flow


slowly

Ion exchange chromatography


separation by charge
Beads have charged group:
+ charge binds acidic amino acids
- charge binds basic amino acid
Different proteins bind with different affinity
Eluted with increasing amount of salt (NaCl or KCl)
Different proteins elute at different salt
concentrations

KROMATOGRAFI AFINITAS KOLOM Ni

VEKTOR
EKSPRESI
Senyawa penginduksi :
IPTG (analog laktosa)

PROTEIN
FUSI
Pada plasmid pET
16b :

HisHisHisHisHis
His
(His-Tag)
His-Tag

Ssisi
restriks
i Faktor
Xa

Protein target

: untuk pemurnian dengan kolom


afinitas Ni2+

Sisi restriksi Faktor Xa


: sisi pemotongan yang
dikenali oleh Faktor Xa yang akan
memotong setelah arginin dari Gly-Arg
dengan tujuan untuk mendapatkan
protein target murni tanpa
His-Tag
Protein target : hasil ekspresi dari gen yang diklon
ke dalam vektor
ekspresi yang
digunakan

Fusion Proteins-how they are made and recovered

DNA
peptide
mRNA

Recover from
culture medium
and purify

protein of
interest
Maltose-binding protein

Fusion Proteins

KROMATOGRAFI KOLOM
HODROFOBIK
Protein mempunyai daerah
hidrofobik di permukaan
yang dapat berinteraksi
dengan resin
Resin yang mengandung
gugus fenil atau alifatik
mengikat protein : protein
yang paling hidrofobik akan
terikat paling kuat
Elusidilakukan dengan
pelarut yang kurang polar
atau dengan menurunkan
garam

PENGUKURAN
KONSENTRASI PROTEIN
Absorbansi pada 280 nm
Bradford assay
Lowry assay
Bicinchonicic acid (BCA) assay

Verification

SDS PAGE (Sodium dodecyl sulfate


polyacrylamide gel electrophoresis) is used for
verify target protein.

Polymerization :
- polymer of acrylamine/bisacrylamide
- TEMED, ammonium persulfate
catalyst for polymerization

SDS-PAGE
1. Heat sample with SDS and mercaptoethanol
SDS = Detergent (anionic)
- Denatures proteins
- Coats proteins
- Each protein has
similar mass/charge
ratio
-mercaptoethanol/DTT :

reduces disulfide bonds


2. Separate on polyacrylamide gel
Protein migrates through gel
matrix in electric field.

Protein visualization on gels


Immediately after electrophoresis proteins in the gels
are precipitated by either adding alcohol containing
solutions or strong acids (e.g. TCA).
Protein are often stained by Coomassie Blue dye or
by photography-like treatment with AgNO3 (silver
staining)

Protein separation using SDS-PAGE


(Laemmli system)

1. Apply protein/dye samples


into polyacrylamide gel wells

Stacking
gel

Resolving
gel

3. Remove the gel from the


apparatus and stain for
proteins

2. Run the electrophoresis until dye


reaches the end of the gel

MIGRASI PROTEIN PADA GEL AKRILAMID

Example of silver stained gel


Silver staining is usually
10-100 times more
sensitive than Coomassie
Blue staining, but it is
more complicated.
Faint but still visible bands
on this gel contain less
than 0.5 ng of protein!

SDS PAGE of Purification

1.
2.
3.
4.
5.

Complete mix of proteins


High Salt
Ion exchange
Gel-filtration
Affinity

10 micrograms loaded in each lane

CONTOH HASIL SDS PAGE


SDS PAGE

94
67
43
30
20
14
kDa

KETERANGAN
1.

Marka protein

2.

Protein Fraksi 1

3.

Protein fraksi 2

4
3

NATIVE PAGE

Disebut juga Non- denaturating PAGE

Tanpa penambahan SDS dan mercaptoethanol

Protein tidak terdenaturasi

CONTOH HASIL NATIVE PAGE

Electrophoresis :
Iso-electric focusing (IEF)

IEF : identify two similar proteins


that are difficult to separate by
any other means
Separates proteins by their
isoelectric points (pI)
Each protein has own pI = pH at
which the protein has equal
amount of positive and negative
charges of charged amino acid
(the net charge is zero)
IEF 4-6.5 pH
gradient

IEF
Mixtures of ampholytes,
small amphoteric molecules
with high buffering capacity
near their pI, are used to
generate the pH gradient.
Positively and negatively
charged proteins move to
and +, respectively, until they
reach pI.
PI of proteins can be
theoretically predicted.
Therefore, IEF can also be
used for protein identification.

2D PAGE
Two dimensional electrophoresis, which separates
proteins based on their electrical charge (IEF) and size
(SDS PAGE)

Analytical methods

HPLC systems use greater pressure to force the


extract through the column in a shorter time
Limitations : Less protein is separated

Mass spectrometry : suspend the sample into a


gas phase separated molecules based on their
mass-to-charge ratios detect separated ions
the identity and size of most protein fragments

DOT BLOT

CONTOH HASIL DOT BLOT

Keterangan:
1.

Protein fraksi 1

A.

Serum preimun

2.

Protein fraksi 2

B.

Serum imun primer

C.

Serum booster ke-1

D.

Serum booster ke-2

E.

Serum anti MAP

Western Blotting (WB)


WB is a protein detection technique that combines
the separation power of SDS PAGE together with
high recognition specificity of antibodies
An antibody against the target protein could be
purified from serum of animals (mice, rabbits, goats)
immunized with this protein
Alternatively, if protein contains a commonly used
tag or epitope, an antibody against the tag/epitope
could be purchase from a commercial source (e.g.
anti-6 His antibody)

WESTERN BLOT
warna ungu
AP

Protein ditransfer
ke membran
SDS-PAGE
nitroselulosa

+ Nitro blue tetrazolium (NBT)


+ 5-Bromo-4-Chloro-3-indolyl phospate (BCIP)

Ab 2o
Ab 2o
Ab 1o
Protein target
Membran nitroselulosa

Dilabel dengan Visualisasi


antibodi spesifik

Contoh hasil Western blot

WB: 4 steps
1. Separation of proteins using SDS PAGE
2. Transfer of the proteins onto e.g. a
nitrocellulose membrane (blotting)
3. Immune reactions
4. Visualization

WB : Blotting

WB :Detection

CONTOH HASIL WESTERN BLOT


Western blot

SDS PAGE

KETERANGAN:

94
67

1.

Marker protein

2.

Protein Non
induksi murni

30

3.

Protein Induksi
murni

20

4.

Ekstrak protein
total

43

14
kDa

Ab 10= 1:
2500

MS-MALDITOF
= MASS SPECTROPHOTOMETRY- MATRIX ASSISTED LASER
DESORPTION IONIZATION TIME OF FLIGHT
Digunakan untuk konfirmasi kebenaran suatu protein
Pencarian protein yang homolog dilakukan menggunakan program
komputer MS/MS Ion Search dari Mascot (
http://www.matrixscience.com)
Tahapan:
Isolasi protein target dari gel
Proteolisis dengan enzim tripsin hingga diperoleh fragmen
peptida
Ekstraksi dengan asetonitril
Ekstrak dikeringkan dan diresuspensi dalam larutan matrix
Larutan digunakan dalam MS-MALDITOF
Proteomics international Australia

SPEKTRUM HASIL MALDITOF

HOMOLOGI PROTEIN
No
.

Nama Protein

1.
2.
3.
4.
5.
6.

Ornithine carbamoyltransferase
AE006587 NID
AE010070 NID
AE007317 NID
AF534569 NID
Ornithine carbamoyltransferase

7.
8.
9.
10.

Ornithine carbamoyltransferase
Ornithine carbamoyltransferase
Ornithine carbamoyltransferase
Ornithine carbamoyltransferase

Sumber
S. pyogenes
S. pyogenes M1 GAS
S. pyogenes MGAS8232
S. pneumoniae R6
S. gordonii
S. pneumoniae (galur
TIGR4)
P. aeruginosa
P. mendocina
S. typhi
S. typhimurium

Ukuran
(kDa)
37,7
37,9
37,8
38,0
37,8
37,8
37,9
37,8
36,5
36,5

Preserving Proteins

One means of preserving protein is


lyophilization or freeze drying.

Methods : the protein is first frozen a


vacuum used to hasten the evaporation of
ice water crystals (sublimation) the
containers are sealed may be stored at
room temperature for prolonged periods
of time