Protein Purification:

From industrial enzymes to cancer therapy

1 1
 Protein Expression and Purification
Ekspresi Protein dan
Rangkaian Pemurnian Series
pengantar Introduction
ekspresi protein Recombinant protein expression and
rekombinan dan
purification for biomanufacturing
pemurnian untuk
biomanufacturing Dihydrofolate reductase
dihidrofolat reduktase Affinity purification
pemurnian afinitas
Perform affinity chromatography
Melakukan
kromatografi afinitas Perform size exclusion (desalting)
melakukan eksklusi chromatography
ukuran (desalting)
Quantitate purified protein
kromatografi
Menghitung protein yg
dimurnikan 2
 The production of
pharmaceutical proteins using
genetically engineered cells
Biomanufacturing
Defined Produksi protein farmasi
menggunakan teknik
rekayasa genetika

3
 Expression Choices
Pemilihan Ekspresi
Cell type:
E. coli
Yeast
Mammalia

Tipe sel :
E. coli
Ragi
Mammalia
4
 Expression Choices
Parameter Bacteria Yeast Mammalian

Risiko kontaminasi Low Low High

Cost of growth Low Low High

medium

Product titer High High Low

(concentration)

Folding Sometimes Probably Yes

Glycosylation No Yes, but different pattern Full

Relative ease to grow Easy Easy Difficult

Relative ease of Difficult Easy Easy

recovery
Deposition of product Intracellular Intracellular or extracellular Extracellular

Product Intracellular Often secreted into media Secreted

5
PROTEIN: USED IN THE Cell
TREATMENT OF: Production
Insulin Diabetes E. coli
Human growth hormone Growth disorders E. coli
Granulocyte colony stimulating factor Cancers E. Coli
Erythropoietin Anemia CHO cells
Tissue plasminogen activator Heart attack CHO cells
Hepatitis B virus vaccine Vaccination Yeast
Human papillomavirus vaccine Vaccination Yeast

Chinese hamster ovary (CHO) cells

6
 Converts dihydrofolate into tetrahydrofolate (THF)
by the addition of a hydride from NADPH

THF is a methyl (CH3) group shuttle required for

DHFR synthesis of essential molecules
Dihydrofolate - nucleotides
reductase - amino acids

Mengkonversi dihidrofolat menjadi tetrahidrofolat (THF)

dengan penambahan hidrida dari NADPH

THF adalah kelompok metil (CH3) yg diperlukan untuk

sintesis molekul penting
- nukleotida
- asam amino

7
 DHFR inhibition or reduction disrupts nucleic acid
synthesis affecting
-Cell growth
-Proliferation

DHFR and Cancer

Methotrexate one of the first chemotherapeutic agents
-Inhibits DHFR
-Methotrexate resistance - correlates with
amplification of DHFR genes

penghambatan DHFR atau pengurangan mengganggu

sintesis asam nukleat yang mempengaruhi :
-Pertumbuhan sel
-Proliferasi

Methotrexate - salah satu agen kemoterapi pertama

-Menghambat DHFR
-resistensi Methotrexate - berkorelasi dengan amplifikasi gen
DHFR

8
GST-DHFR-His Construct
GST DHFR - His

Glutathione-s-transferase
Added to increase solubility (ditambahkan utk meningkatkan
kelarutan)
Can be used as a secondary purification methodology (dpt
digunakan sbg metode pemurniak kedua)

Tanda Histidine
6 Histidine tag that binds to certain metals such as nickel
6 histidin menandakan adanya ikatan khusus dg metal
misalnya nikel

Human dihydrofolate reductase

Gene product of interest (produksi gen
penting)
Target for chemotherapy reagents (target
9
reagen kemoterapi)
 Transcriptional
Regulation in the
pDHFR system

10
growth
Cell

11
 Recovery
Separation of protein from other molecules

Purification
Separation of the protein of interest from other
Protein GST- proteins
DHFR-His
Pemulihan
Pemisahan protein dari molekul lain

Pemurnian
Pemisahan protein penting dari protein lainnya

12
Protein Expression and Purification Series
(sel yg sdh ditandai) Streak Cells

(kultur didiamkan semalam) Overnight culture

Subculture, monitor, and induce

(hasil dan lsel lisis) Harvest and lyse cells

Purify (pemurnian)
Centrifugation or Instrumentation

Analyze (analisis)
13
 Mobile phase (solvent and the molecules to be separated)

Stationary phase (through which the mobile phase travels)

paper (in paper chromatography)
glass, resin, or ceramic beads (in column chromatography)

Molecules travel through the stationary phase at different rates because of their chemistry.

fase gerak (pelarut dan molekul untuk dipisahkan)

fase stasioner
kertas (dalam kromatografi kertas)
kaca, resin, atau manik-manik keramik (di kromatografi kolom)
Molekul berjalan melalui fase diam pada tingkat yang berbeda karena sifat kimia mereka

14
 Ion Exchange (protein charge)

Size Exclusion (separates on size)

Hydrophobic Interaction (hydrophobicity)

Affinity:
Protein A
His-tagged
Glutathione-s-transferase

Types of Column Penukar Ion

Chromatography Size Exclusion(memisahkan ukuran)
Interaksi hidrofobik (hidrofobik)
Afinitas:
Tipe kromatografi protein A
Kolom histidine
Glutathione-s-transferase

15
Pour column
Wash resin to remove
Affinity purification
packing buffer
Equilibrate resin
Bind GST-DHFR-His
Elute unbound proteins
Wash protein bound onto
the resin
Elute GST-DHFR-His
tuangkan kolom
Cuci resin untuk
menghilangkan bungkus
penyangga
menyeimbangkan resin
Mengikat GST-DHFR-His
Elusi protein yg tdk terikat
Cuci protein terikat ke
resin
Elusi GST-DHFR-His
Pouring a 100 l Ni-IMAC column
Label column with initials. Prepare
column. Snap off bottom tab of empty
column, remove cap and place in 2 ml
collection tube.
Tandai kolom dengan inisial. Siapkan
kolom. Snap off tab bawah kolom
kosong, hilangkan cap dan tempatkan
di 2 ml tabung koleksi (ependrop?)
200 l
Ni-IMAC
Add 200 l of Ni-IMAC resin
slurry to empty column resin
Tambahkan 200 l Ni-IMAC slurry
resin slurry ke kolom yg
kosong
Centrifuge for 2 minutes at 1,000 x
g. After spin, discard buffer that has
collected in the collection tube.
Centrifuge selama 2 menit pada
1.000 x g. Setelah itu hilangkan
buffer yang telah didapat
16
 Affinity purification
Washing and equilibrating the 100 l Ni-IMAC column

200 l

Add 200 l of distilled Distilled

Pour column H2O to column H2O
Wash resin to
remove packing
buffer Centrifuge for 2 minutes at
1,000 x g. After spin, discard
Equilibrate resin
water from collection tube.
Bind GST-DHFR-His
500 l
Elute unbound proteins Add 500 l of Equilibration
Equilibration
Wash protein bound buffer to column
buffer
onto the resin
Elute GST-DHFR-His Centrifuge for 2 minutes at
1,000 x g. After spin, discard
Equilibration buffer and
collection tube. The column is
now ready to use. 17
 Affinity purification
Binding the GST-DHFR-His to the Ni-IMAC resin

600 l

Place yellow tip closure on bottom

Pour column of column. Add 600 l Soluble Soluble fraction
Wash resin to remove Fraction to Column; Put on clear
top cap.
packing buffer
Tempatkan yellow tip di bawah
Equilibrate resin kolom. Tambahkan 600 ml Fraksi
larut ke Kolom lalu ditutup
Bind GST-DHFR-His
Elute unbound proteins
Wash protein bound Gently mix for 20 min.
onto the resin Campur dgn hati2 20 menit

Elute GST-DHFR-His

18
 His tags
His tags are typically a
series of 6 histidines
His tag and column interaction
added
Histidine
-OOC N3H+
to the C or N terminus of a
recombinantNi protein

Resin

His-tagged
Recombinant
Protein

19
 His tags
His and imidazole structure similarities
Imidazole competes with His for Ni2+ sites

Histidine Imidazole
-OOC N3H+

20
 Affinity purification
Performing affinity chromatography

Label three 2 ml tubes:

Flow through, Wash and Eluate. Flow
through
fraction
Pour column Remove yellow tip closure. Place
column in 2 ml collection tube
Wash resin to remove labeled Flow Through and remove
packing buffer clear top cap. Centrifuge for 2 min at
1,000 x g.
Equilibrate resin
Bind GST-DHFR-His Set aside Flow Through.
600 l
Elute unbound
proteins Place column in 2 ml collection tube Wash Wash
labeled Wash. Add 600 l Wash fraction Buffer
Wash protein bound Buffer to column.
onto the resin
Elute GST-DHFR-His
Centrifuge for 2 min at 1,000 x g.

Set aside Wash fraction.

21
 Affinity purification
Performing affinity chromatography (continued)

400 l
Place column in 2 ml collection
Pour column tube labeled Eluate. Add 400 Elution
Eluate
l Elution Buffer to column. Buffer
Wash resin to remove
packing buffer
Equilibrate resin
Bind GST-DHFR-His Centrifuge for 2 min at 1,000 x g.

Elute unbound proteins Set aside Eluate.

Wash protein bound onto
the resin
Elute GST-DHFR-His Collected fractions

Flow through Wash Eluate

22
~600 l ~600 l ~400 l
Size exclusion purification
(buffer exchange)
Eluate GST-DHFR-His in 20 mM sodium
fraction phosphate, 300 mM NaCl and
250 mM imidazole

Imidazole

250 mM
imidazole
W and Y contribute to
solution has
A280 of proteins
an A280= 0.2-0.4

NEED TO REMOVE IMIDAZOLE TO QUANTIFY

PROTEIN CONCENTRATION USING A280 23
 Size
Exclusion

24
Protein analysis (Quantitation using A 280)

75 l
Clean UV
Desalted cuvette
eluate

Set absorbance to 280 nm

Blank spec with distilled H2O

Measure absorbance of sample at 280nm

Atur absorbansi pd 280nm

Blanko diisi distilled H2O
Ukur sample pd 280nm

25
Enzyme Assay

26