BIOTEKNOLOGI FARMASI

Karakterisasi Protein
Rekombinan

apt. Malinda Prihantini, M.Si.

apt. Danang N. Wibowo, M.Farm.

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 PRODUK REKOMBINAN

PROSES HULU PRODUKSI PROSES HILIR

(up stream) (Fermentasi) (down stream)

• Isolasi gen • Batch • Pemisahan

target • Fed-batch Sel
• Konstruksi • Continuous • Lisis sel
vektor batch • Isolasi Protein
rekombinan • Pemekatan
• Transformasi protein
ke sel inang • Pemurnian
• Seleksi klon • Karakterisasi
• Formulasi

Bioteknologi Farmasi Semester V & VII ISOLASI DAN Protein

PURIFIKASI PROTEIN
FAC1D5W10 Karakterisasi Rekombinan
Mengapa karakterisasi harus dilakukan ?
 Karakter Protein
• Struktur protein
• Monomer, homo-mer / hetero-mer
• Modifikasi /konjugasi
• (glikoprotein, lipoprotein, ribonukleoprotein, PEGilasi-
polietilen glikol
• Domain/daerah ikatan spesifik (Ag – Ab,
protein pengikat
• Dua dimensi (-heliks, -sheet)
• Globul / friber

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 Karakter protein
• Urutan protein
• Situs pemotongan protease
• Ukuran protein
• Panjang asam amino
• Bobot molekul (kDa)  http://web.expasy.org/compute_pi/
• Muatan protein (pI)  http://web.expasy.org/compute_pi/
• Hidrofilisitas/hidrofobisitas  http://web.expasy.org/protscale
Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan
 Karakter Protein

• Stabilitas (pH, T, tekanan,dll)

• Aktivitas biologis
• enzimatik (substrat  produk)
• proteolisis, hemolisis dll

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 Struktur protein

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan
 Sisi pemotongan protein

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan
Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan
 Karakterisasi Protein
• Metode karakterisasi tergantung pada karakter
protein target
• Karakter protein  sudah diketahui / belum ?
• Jika sudah diketahui  uji untuk konfirmasi
identitas / karakter protein
• Jika belum diketahui  uji untuk mengetahui
karakter (karakterisasi)

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 Karakterisasi Protein
• Identitas protein
• Kemurnian
• Cemaran
• Jumlah/konsentrasi protein
• Stabilitas/penyimpanan/sterilitas
• Aktivitas
• Spesifisitas/reaksi silang
Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan
 Karakterisasi Protein
1. Purity and Impurities  modifikasi ??
– Glikosilasi, ik. Disulfida (alteratif), deamidasi,
oksidasi, fosforilasi, asetilasi, sulfasi, sulfoksidasi,
gamma-karboksilasi, pyroglutamat.
– Proses produksi protein rekombinan: vector,
inang, medium pertumbuhan, teknik isolasi
– Aktivitas , toksisitas
– Peptide mapping, MS, SDS PAGE, RP-HPLC
Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan
 Karakterisasi Protein

• Identity  konfirmasi komposisi molekular

• Posttranslational modification
• Amino acid analysis, Peptide mapping, MALDI-MS,
ESI – MS (electrospray ionization), koefisien
extinsi, pI, struktur kristal, NMR, spektrum
CD(circular dichroism), HPLC, CE, PAGE

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 Karakterisasi Protein
• Penentuan Jumlah / Kuantifikasi Protein
• OD, kolorimetri (Bradford, Lowry, BCA-Bicinchoninic,
biuret),Kjeldahl, Bioassay, Immunoassay
• Stabilitas, Penyimpanan dan Sterilitas
• Mempengaruhi kualitas, keamanan dan efikasi.
• Fisikokimia, biologi, mikrobiologi
• Spesifisitas dan Cross-reactivity
• Produk antibodi
• Uji pengikatan, epitope mapping/kabohidrat
• In vitro +  in vivo
Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan
 Metode Karakterisasi
Protein

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 PENENTUAN KONSENTRASI PROTEIN
1. UV spectrophotometry Absorbance at 280nm wave lenght
Absorbance of aromatic amino acid, i.e. tryptophan, tyrosine, phenylalanine
Protein with no or less aromatic residue ?
2. Colorimetry  absorbance at visible light 400 –
700 nm
* Lowry Method : Biuret reaction : Protein
(Nitrogen) + copper (II) under alkaline condition can
reduced Folin Ciocalteu
phosphomolybdicphosphotungstic acid 
heteropolymolybdenum blue

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

KARAKTERISASI STRUKTUR
PRIMER / URUTAN PROTEIN
• N-terminal sequencing (10-15 first
amino acids) – Edman degradation
• C-terminal sequencing – carboxy
peptidase
• Peptide mapping - using protease  LC-
MS/MS

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

Amino terminal Carboxy
Sequencing terminal
Sequencing

N C

Peptide mapping

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 PROTEIN SEQUENCING

• IDENTITY

• HOMOGENITY (MAB)

• PROTEOLITIC PRODUCT

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 PEPTIDE MAPPING

• IDENTITY
• CONFIRMATION OF GENETIC STABILITY
• CONSISTENCY OF LOT PRODUCTION
• GLYCOSYLATED PEPTIDE (MAB)
• WRONG AMINO ACID (1 OR >1)

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 Analisis asam amino

• Penentuan komposisi asam amino suatu

protein
• Identitas, kontaminan, modifikasi

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 KARAKTERISASI PROTEIN

BERDASARKAN:
1. UKURAN  SDS-PAGE
2. MUATAN  ISOELEKTRIC FOCUSING
3. IMMUNOASSAY  Western blot/ ELISA

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 SDS - PAGE
 Protein is denatured – SDS (sodium dodecyl sulphate)
  to obtain protein which is homogeneous
structurally (linier)
 protein is negatively charge
 in electric field : protein migrate to positive (anode)
 migration based on protein molecular weight (size)
 Porous matrix  acrylamide gel

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 SDS ACTION ON PROTEIN

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 RESOLUTION OF ACRYLAMIDE GEL
PERCENTAGE OF SEPARATION
ACRYLAMIDE CAPACITY

15% 15 – 45 kDa

12,5% 15 – 50 kDa
10% 18 – 75 kDa

7,5% 30 – 120 kDa

5% 60 – 212 kDa
Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan
 PREPARATION OF SDS PAGE
A. separating gel

B. stacking gel

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 SDS-PAGE CHAMBER

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

EXAMPLE OF SDS – PAGE RESULT
205 kDa
116 kDa
97 kDa
66 kDa
45 kDa
29 kDa

Coomassie blue stainig Silver staining

Silver : 0,5 - 1,2 ng
Limit of detection : Coomassie : 36 - 47 ng

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 NATIVE PAGE

 WITHOUT SDS TREATMENT

 PROTEIN IS NOT DENATURED

 NON-DENATURING PAGE

 PROTEIN SEPARATION BASED ON NATIVE CONFORMATION

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

PERBEDAAN SDS PAGE & NATIVE PAGE

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 ISOELECTRIC FOCUSING

 Protein characterization based on total electric charge

 using acrylamide gel
 electrophoresis is done by pH gradient between cathode
and anode in ampholyte solution
 pI (isoelectric point) : pH total electric charge of a
protein is zero  protein will not migrate in electric
field

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 ISOELECTRIC FOCUSING

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan
Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan
Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan
Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan
 TWO DIMENSIONAL (2D)
ELECTROPHORESIS
 Combination of IEF & SDS-PAGE
 To characterize / differentiate protein which have
the same molecular weight but different in electric
charge
 To confirm purity of a protein
 First run : isoelectric focusing
 Second run : SDS-PAGE
Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan
Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan
Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan
 Maldi-Tof / Mass Spect / LC MS-MS

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan

 WESTERN BLOT
 Protein characterization based on immune reaction Antigen –
Antibody
 Able to detect a protein in a mixture
 Could get the information of protein size (rainbow protein marker )
 Specific antibody against target protein is needed (as
primary antibody)

 Secondary antibody  conjugated with an enzyme for visualization

 i.e. : horse radish peroxidase (HRP)
alkaline phosphatase (AP)
Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan
Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan
 ANY
QUESTION…?

Bioteknologi Farmasi Semester V & VII Karakterisasi Protein Rekombinan