Professional Documents
Culture Documents
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
Insect system
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
SENYAWA PENGINDUKSI
RNA
polymerase
promoter
operator
gene1
gene2
gene3
gene4
DNA
repressor
repressor protein
RNA
polymerase
RNA repressor
TATA
polymerase
gene1
induces transcription
gene2
gene3
gene4
repressor
promoter
operator
DNA
repressor protein
lactose
repressor
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
PROTEIN EKSTRASELULER
PEMECAHAN SEL
PEMISAHAN SEL
PEMEKATAN PROTEIN
PEMEKATAN PROTEIN
PEMURNIAN PROTEIN
LOKASI PROTEIN
PROTEIN INTRASELULER :
Sitoplasma
Periplasma
PROTEIN EKSTRASELULER :
medium
KET :
PROTEIN
SEL
SENTRIFUGA
SUPERNATAN DIAMBIL
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
Cell lysis
HOMOGENIZER
SONICATOR
GLASS BEAD
HOMOGENIZER
Protein precipitation
Filtration (sized based) separations
methods
Chromatography
Electrophoresis
Analytical methods
Protein precipitation
Strategi pemurnian
Muatan : kromatografi penukar ion
Kepolaran : kromatografi in teraksi hidrofobik
Ukuran : dialisis, ultrafiltrasi, gel elektroforesis,
kromatografi filtrasi gel, dan ultrasentrifugasi
Spesifitas : kromatografi afinitas
Membrane filtration
Dialysis
Diafiltration
Column chromatography
Proteinmixtureappliedtocolumn
Solvent(buffer)appliedtotop,flowed
throughcolumn
Differentproteinsinteractwithmatrixto
differentextents,flowatdifferentrates
Proteinscollectedseparatelyindifferent
fractions
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
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
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 :
Stacking
gel
Resolving
gel
1.
2.
3.
4.
5.
94
67
43
30
20
14
kDa
KETERANGAN
1.
Marka protein
2.
Protein Fraksi 1
3.
Protein fraksi 2
4
3
NATIVE PAGE
Electrophoresis :
Iso-electric focusing (IEF)
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
DOT BLOT
Keterangan:
1.
Protein fraksi 1
A.
Serum preimun
2.
Protein fraksi 2
B.
C.
D.
E.
WESTERN BLOT
warna ungu
AP
Protein ditransfer
ke membran
SDS-PAGE
nitroselulosa
Ab 2o
Ab 2o
Ab 1o
Protein target
Membran nitroselulosa
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
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
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