Tạo dòng, biểu hiện và tinh sạch protein hGM CSF (human granulocyte macrophage colony stimulating factor) trong chu chất tế bào escherichia coli

Science & Technology Development, Vol 17, Ne.T4-2014

Tao clang, bi4u hiOn va tinh sach
protein hGM-CSF (Human Granulocyte
- Macrophage Colony - Stimulating
Factor) trong chu chat to bao
Escherichia coli
• Nguyen Phu Sang
• Nguyen Thanh Thao
• Tran Van Hieu
Tru,o,ng Dal hoc Khoa hoc Ty' nhien, DHQG - HCM
( Ben nkin ngay 30 thong 10 ndm 2014, nhdn dang ngay 16 (hang 01 nam 2015)

TOM TAT
hGM-CSF /à met cytokine bao gem 127
amino acid, vol 4 vj tri cystein hinh thanh 2
cau n6i disulfide. Mac de, dang tw nhien cOa
hGM-CSF la glycoprotein nhung stir glycosyl
h6a kh6ng c6 anh huerng den chirc nang
sinh h9c caa protein nay. GM-CSF c6 cherc
nang kith thich sw ten tai, tang sinh, biet h6a
cOa cac to bao tien than No mau va tang
ctrang chuv nang c0a cac to bac, mau
twang thanh. GM-CSF (15 dut;rc sur dyng
rang rai de diet, trj nhieu loci benh nhu':
chYng suy giam bpch cau trung tinh do hea
tri ung thy se, dyng trong coy ghep fay
xtrang, benh suy giam bpch ceu tOy cep...
Trong nghien cOv nay thong tai trinh bay kat
qua too dang va bleu hien protein hGM-CSF
trong chu chat to bac) Escherichia coli. Gen

ma h6a cho GM-CSF ngtrai 'Ivey nhan ban
bang phtrang phap PCR voi cap mei CO
mang trinh tu' cat giol hpn cOa Ncol va Xhol.
Dopn DNA khuech dpi cluvc dang h6a thanh
tong vat) vi tri Ncol/Xhol fret) vector pET-

22b va dyer chen clOng khung vai trinh
tin MO tiet pelB. Vector bleu hien pET-hGM
duer bien nap vac) to bao E. coli 8L21(DE3)
va the bien nap dtrac cam Ong IPTG de
kiem tra kha nang bleu hien protein hGMCSF. Phan dopn protein chu chef duix thu
nhan bang phuang phap sec them than. Sir
bleu hien cOa hGM-CSF duet kiem tra bang
dien di SDS-PAGE cac phan Clopn protein
king, to bac) chef va chu chef Protein trong
phan dopn chu chat sau (96 &rot tinh the
bang sec ky trao del anion sir dyng cat Q
Sepharose FF voi phyang phap dung ly tang
nang do mu6i. Ket qua dien di SDS-PAGE
cho they c6 sw hien dien vpch protein twang
dvang vai hGM-CSF d pha chu chef cua
thong E. coli BL21(DE3)/pET-hGM va
protein thu nhan claw qua boat sec V dat
de tinh sach 97,4 %. Ket qua bieu hien va
tinh sach tren da dyer khang djnh bang
phvang phap Western Blot II& khang the
dac hieu khang hGM-CSF.

Ter kh6a: GM-CSF, chu chef, sec ky trao d6i anion

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TAP CHI PHAT TRItti KRIM TAP 17, SO T4-2014

Mlle DAU
Nhan to kich thich tao khem bach cau hat-dai
thuc bao 6 nguai — hGM-CSF (human
Colony-Stimulating
Granulocyte-Macrophage
Factor) — la met cytokin ce ban chat la
glycoprotein, gem 127 amino acid c6 tong lueng
phan tir tir 14 den 35 kDa tit), theo tinh tang
dui:mg hea. GM-CSF oft hai vj tri N-glycosyl hea
va ben vj tri 0-glycosyl hea, tuy nhien su
glycosyl hea khong anh twang den hoot tinh sinh
hoc cia protein nay [1]. GM-CSF CO hai cau nei
disulfide net phan to hinh thanh gitta cite gec
a not
Cys54 va Cys96, Cys88 va Cys121. Cu
disulfide the nhat (54-96) lien quan den hoot tinh
sinh hoc cia GM-CSF trong khi cau not disulfide
con lai khong co firth hue:mg tort hoat tinh [2].
GM-CSF nguiri tai to hqp da duqc sir dung rang
Ili de dieu tri cac benh nhu suy giam bach cau
trung tinh, benh bach eau toy cap, sir dung trong
city ghep to bao g6c tao mau hay lam to duqc
vac-xin. Cho den nay, cytokin nay can duqc chi
dinh cho clic benh nhan bj suy giam mien djch,
AIDS hay bj nhiem Ming... [3, 4]. Nhu vay, vii

tiem nang ling dung ceng them nhu cau sir dung
ngay met nhieu, hGM-CSF la met trong nlang
protein tit to hqp dang duqc quan tam nghien
ceu va san xuat hien nay.Trong cac he thong
duqc sir dung a bleu hien protein tai to hqp thi
E. toll IA met he thong duqc sir dung kha phe
bieu. Protein tai to hqp ce the duqc bieu hien 6
cac dang khac nhau va trong cac khong gian khac
nhau a to bao E. colt. t3 day thong toi lua chon
tiet vio chu chit vi no co nheng uu diem phO hqp
vei protein muc tieu nhu san pham tao thanh
thuerng a dang tan va khong chira methionine dau
N, vOng chu chat tao mot truang thuan Iqi cho
viec hinh thanh cau not disulfide [5, 6].
Trong Ink bio nay, chimg toi trinh bay cac
ket qui deng hea va bieu hien hGM-CSF trong
chu chat to bio E. colt nhtr la buOc nen tang ban
din trong quy trinh nghien cOu nham san xuAt ra
protein nay.

VAT LBW VA PHUVING PliAP
Chiang vi sinh vat va plasmid
endA 1
Chitng vi khuan E. colt DH5a
hsdR17 (rk-/mk) supE44 thi X recAl gyrA96
AlacU169 (080 lacZ cIM15)] (Takara) duqc sir
dung de tao deng va km trft plasmid. E. colt
ompT, hsdS (rr mB), gal (DE3)]
BL21(DE3)
duqc sir dung lam chOng chit de bieu hien protein

muc tieu.
Plasmid pET-22b (Novagen), kich thtrec
5493 by china promoter T7 duqc cam Ong bang
IPTG(isopropyl I3-D thiogalactoside), gen khang
ampicillin (Amp) duqc ding de tao plasmid pEThGM bieu hien hGM-CSF. Plasmid pET-22b con
co met trinh to tiet pelB 0 dau N, khi gen muc
tieu &cm then deng khung yeti trinh to nay,
protein duqc tao thimh se duqc tiet vio vimg chu
chat to No E. colt.
Tich chit plasmid
Cac plasmid duqc tech chi& theo phtrong
phap SDS-kiem theo mo to cita Sambrook va
Russell [7].
Thiet kg plasmid tai to Ito plET-hGM mang
gen hgm-csf ml boa cho protein hGM-CSF
Gen hgm-csf ma hea cho protein hGM-CSF
duqc thu nhan tir plasmid pGM-CSF (cung cap
ben BM. Cong nghe Sinh hoc Phan tO - Mai
outing, Tnrang Doi hoc Khoa hoc To nhien
TPHCM) bang each PCR vOi hai mit 5'-Nco va
3'-Xho theo chtrang trinh 95 °C15 phut; 30 chu
ky: 94 °C/45 phid, 55 °C/45 giay, 72 °C/45 phitt;
ket thec, 72 °C/10 phut. Hai mei nay se bat cap
vao khuon va nhan ban doan trinh to hgm-csf.
San phiun tao ra la doan gen co kich thudrc
khoing 415 by cling vont vi tri cat gioi hart cOa
enzyme Xhol va Ncol 6 hai dau. San pham PCR
duqc xti ly tao dau dinh vei hai enzyme cat gioi
hen tren va tinh the bang EZ-10 Spin Column
DNA Gel. Extraction kit (BioBasic, Canada) theo

huang din cOa nha san xuAt
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Science & Technology Development, Vol 17, No.T 4-2014

Plasmid pET-22b cling duck; cat ma yang bang
cap enzyme Xhol vi Ncol, bet hoat enzyme bang
phenol/chloroform va tinh che bing EZ-10 Spin
Column PCR Products Purification kit (BioBasic,
Canada). Kich thuot cua doan DNA mang gen
hgm-csf va plasmid pET-22b da cat mer yang
dirge xac dinh bing dien di tren gel agarose 1 %.
Plasmid pET-hGM dime eau tree bang each
A.

not gen hgm-csf da xu ly veri Xhol va Ncol vao

plasmid pET-22b cling dtrac xu ly yeti hai
enzyme nay.
Tao to bio khi nap va Min nap plasmid pEThGM vio to bao E. coliDH5a
Tao to bao E. coli kha nap bang phuang phap
calcium lanh vi pET-hGM dirge bien nap vao E.
coil DH5a bang phuang phi!) sec nhiet theo mo
to caa Sambrook va Russell [7]. Huyen phis to
bao sau bien nap dime trii tren moi truing LB
agar (tryptone 1%; yeast extract 0,5 %; NaCI 0,5
%; agar 2 %) be sung Amp dat ming da cuOi 100
pg/mL (LBA-Amp100), IA 37 °C, 14 - 16 girl
Sing loe the bien nap vi kiem tra bing

enzyme eat giai han vi PCR plasmid
Cic the bien nap DH5a mac dtrqc tren m8i
twang LBA-Amp tiep tuc cluac sang be so be
bing phuang phi!) PCR khan lac vii cap mei
5'-Nco va 3'-)Cho. Thu nhen the bien nap cho ket
qua PCR throng tinh va tach chi& plasmid. Sau
do nen Wirth kiem tra plasmid bing phuang phap
PCR vii cap mcii bit cap tren plasmid
T7pro/T7ter (Novagen) vi cat giai han vii hai
enzyme Xhol va Ncol.
Plasmid tai to hap pET-hGM thu nhen sau
cite qua trinh kiem tra dime tien Minh giai tint' to
theo phuang phap Sanger cai tien bed ding ty
Macrogene (Han Quec).
Ket qua giai trinh At duqc phan tich vi so
sink biing phan mem Jellyfish (Lab Valocity).

Trang 14

Bleu hi'fn protein hGM-CSF
Plasmid pET-hGM dime bien nap vao to bao
E. coli BL21(DE3) kha nap. Chung E. coil tii to
hap duac cam Ong bieu hien hGM-CSF bing
nuoi cay lic trong m8i twang LB, be sung
ampicillin 100 .tg/mL a 37 °C, ttic de lic 250
rpm. Khi ODA dat 0,8; be sung IPTG den neng
da cuii IA 0,5 mM; tiep tuc nu& lee 6.30 °C trong
16 h. Tach cac phan doan protein bing phuang
phap sec them that' theo m8 ti dui Ausubel vi
ceng su [8].

Phin tich SDS-PAGE va lai Western
Cac phan doan protein king, to bao chat, chu
chat thu &roc se &me dien di tren gel
polyacrylamide 12,5 % co su hien dien cua SDS
vi so sanh kich thuac vii thang protein chuen.
Protein sau khi dien di dtrac chuyen len ming lai
vi thirc hien lai Western blot yeti khan the chuet
don clang khang hGM-CSF vi phat hien nher
khang the khang IgG cua chuert ceng hop veri
horseradish peroxidase HRP. Hien phim bang be
Kit ECL (Amersham).
Tinh sack protein hGM-CSF hing sic ki trao
doi anion
Do hGM-CSF co pl = 5,5 a pH = 7,5 protein
tii to hap hGM-CSF tich dien am nen phucrng
phap sic kjr trao dcii anion dime sir dung de tinh
che. Dung dich can bing (ddA): NaCI 20 mM-tris
50 mM, pH 7,5. Dung dich dung ly (ddB):
Na2HPO4/NaR2PO4 0,1 M, NaCI 1 M, pH 7,5. He
thong tinh che AKTA FPLC dugc can bang bing
mac. Cat tinh che anion Q SP FF 5 ml (GE
Healthcare) dtrac rira bang mac, can Wing ben
dung dich A. Mk dim nap vio cat yeti Mc de
(long 2 mUphat, tai can bing cat bang dung dich
A. Dung ly theo bee thang veri tic de clang 2
mL/phtit, 20 % dung dich B va 80 % dung dich A
ttrang to vii 100 % B. Thu nhen plan doan
protein tuong ling vii 20 % va 100 % dung dich
B. Phan tich ket qua bing dien di SDS-PAGE va
lai \red kiting the dee hieu khang hGM-CSF.

TAP CHI PHAT TRIO' KWH, TAP 17, S0 T4-2014
KET QUA VA BIN LUAN
ly too deu dinh bang cap enzyme Xhol va Ncol.
San pham khuech dai gen hgm-csf/ XhoI va Ncol
(km tinh the va net vac, plasmid pET-22b da cat
mo yang vai ding cap enzyme XhoI va
(Hinh 1B). HO'n hop san pham not duce bien nap
vao E. coli DH5a Ira sang loc bang mOi truing
LBA-Amp.

Tao vector tii to hqp pET-hGM mang gen
hgm-csf
Gen hgm-csf &roc thu nhan bang phuong
phi!, PCR tir plasmid pGM-CSF cho san pham
co kich thtroc 415bp Mang fmg vai kich Muck du
kien (Hinh 1A). San pliant PCR sau do &roc xtr

WI hp

41$ bp

1 ,4

Binh 1. Thu dan gen hgm-csf va plasmid pET22b. A, Ket qua thu nhan gen hgm-csf dY plasmid
pGM-CSF. 1, Thang chuim DNA 1kb plus; 2, San
pham PCR thu nhim gen hgm-csf hY plasmid
pGM-CSF.B, Ket qua kiem tra plasmid pET-22b.
1, Thang chuan DNA lkb plus; 2, Plasmid thu

titian duce cat bang enzyme XhoI va NcoI

Ifinla 2. Ket qua PCR khuan lac du tuyen. 1, Thang chuen
DNA 1kb plus;2, San pham PCR khuan lac E. coli DH5a
(dad chimg am); 3, Sim pham PCR vdi khu8n DNA la
plasmid pGM-CSF (di chfmg duong); 4, Sim pham
PCRkhutin lac du tuyen

The bien nap moc duck ten mOi trueing nay
dtmc sang Inc so 6'0 bang phuong phip PCR
khan lac vOi cap mOi 5'-Nco/3'-Xho. Ket qua
dien di kiem tra ten gel agarose 1% tren Hinh 2
cho they san pham IChuach dai la mit each co
kich thuerc 415 by Mang Ong vai kich thtrac gen
hgm-csf.
Khuim lac nay duct tech plasmid, tiep tic
kiem tra plasmid tai to hop bang phuong phip
PCR plasmid va cat giOi hon. O Hinh 3A, gieng
5, san pham PCR plasmid pET-hGM v6i cap mOi
5'-Nco/3'-Xho cho they xuet hien Wit vach co
kich think khoang 415 bp, Mang Ong vdri kich
think gen hgm-csf. Nhu vay, trong plasmid tai to
hop dang hem duce co su hien dien dia gen hgmcsf. Phan Ong PCR veri cap mOi bit cap tren

plasmid T7pro/T7ter cho san pham co kich think
678 by (Hinh 3A, gieng 5), bang kich think dorm
DNA mang gen hgm-csf gifta hai mei. Plasmid
tai to hop sau khi &roc kiem tra bang phuong
phip PCR, duce tiep tic kiem tra bang phuong
phip cat gicii han. Hinh 3B gieng 2 va gieng 3

twang fmg vat plasmid pET-22b Ichong mang gen
Ara plasmid tai to hop pET-hGM duos cat mer
yang bang Xbal. Hinh anh tren bin dien di cho
they co su chenh tech kich thuoc gift hai
plasmid nay. Ben canh dO, plasmid nay khi duce
xir ly veri cap enzyme NcoI va XhoI cho hai vach
Mang fmg vOi kich think plasmid pET22b
(5493bp) va gen hgm-csf (415bp) not vao (Hinh
3B, gieng 4). Nhu vay, co the gen hgm-csf
duqc then vao plasmid pET-22b.
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Science & Technology Development, Vol 17, flo.T 4-2014

415 by
bio

Jri

Binh 3. Ket qua kiem tra plasmid bang PCR va cat gieri han. A, Ket qua kiem tra plasmid bang PCR. 1, Thang
chuan DNA 1kb plus;2, San pham PCR plasmid pET-22b v6i cap mei 5'-Nco/3'-Xho; 3, San pham PCR plasmid
pET-22b v6i cap mei T7-pro/T7-ter, 4, San pham PCR plasmid tai to hqp pET-hGM v6i cap moi 5'-Nco/3'-Xho; 5,
San pham PCR plasmid tai to hop pET-hGM vOi cap moi T7-pro/T7-ter.B, Ket qua kiem tra plasmid tai to hop bang
enzyme cat giai han. 1, Thang chuan DNA lkb plus; 2, Plasmid pET-22b cat bang XbaI; 3, Plasmid pET-hGM cat
bang Xbal; 4, Plasmid pET-hGM cat bang XhoI va Ncol
Plasmid sau khi duoc kiem tra bang phuong
phi') PCR va cat gioi han duoc xac dinh c18 chinh
/tic ye met trinh to bang each giai trinh to va so
sinh vai trinh to ly thuyet bang phan mem

Jellyfish. Ket qua cho they có so ding khung
dich ma vai trinh to tin hieu pelB va tuong &mg
100 % so vol trinh to gen da thiet ke dtra tren
ngan hang gen (ket qua khung trinh bay).
Qua cac ket qua phan tich tren cho they
chimg toi da chon ducic (long vi khan E. coli
DH5a mang plasmid tai to hop mong muen pEThGM, plasmid nay duoc bien nap yao E. coil
BL21(DE3) de too chimg E. coil BL2I(DE3)/
pET-hGM bieu hien GM-CSF ngutri tai to hop.

Bien hien protein hGM-CSF trong chu chAt to
bao E. coli BL21(DE3)/pET-hGM
De cam Ong so bieu hien dm gen hgm-csf
trong to bao E. coli BL21(DE3), chimg toi tien
hanh nu8i cay (long to bao nay trong moi truOng
LB long co b6 sung ampicillin neng dO mit 100
ugh/IL WI cam img IPTG Yeti nOng di5 0,5 mM.
Sau khi cam img tir 16-18 h o nhiet cti) 30 °C,
Wirth thu sinh khi to bio. Thoc
chimg toi
hien tech cac phan doan protein bang phtrang
[Map sac them than theo m8 ti caa Ausubel va
gong so. Sau do sir dung phuong phap SDSPAGE de phan tich protein thu duoc trong cac
phan clop.

4. So hen hien hGM-CSF trong E. coli BL21(DE3)/pET-hGM. A, SDS-PAGE; B, Western Blot v6i khang
the dac hieu khang hGM-CSF;1, Thang phan to luting thap; 2, 3, 4: E. coli BL21(DE3)/pET-hGM cam img bang
IPTG (phan doan protein tong, to bit° chat, chu chat); 5, 6, 7 E. coli BL21(DE3)/pET-hGM (phan doan protein tOng,
to bao chat, chu chat); 8, 9, 10 E. coli BL21(DE3) cam img bang IPTG (phan doan protein tOng, to bao chat, chu
chat)

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TAP CHI PHAT TAIEM DM TAP 17, S614-2014

Tren I-linh 4A, clic gieng 5, 6, 7, 8, 9, 10 la
cac nghiem thac dai chang. De kiem tra stn bieu
hien dm protein hGM-CSF sau khi cam img, so
sanh cac vach dm protein yen cac nghieifi thirc
&Si chimg. Tren bin dien di, cac giLg 2, 3, 4 la
mau cac phan doan protein Wong img vOi phfin
doan protein tang so, to bio chat, chu chit clia
chnng E. coil BL21(DE3)/pET-hGM c6 cam *rig
IPTG, chang tai !than thiy 6 mL phan doan chu
chit c8 sir xuat hien mat vach protein nim gifta
vach 14,4 kDa va 20,1 kDa cult thang phan tit
luqng, twang Crag vai kich thuac cna protein
hGM-CSF ly thuyet (gieng 4). Trong khi mL
protein trong cac phan doan chu chat a nghiem
thirc dai chimg (gieng 7, 10) khang nhan thay sv
xuat hien cua vach protein nay. Nhu stay, chimg
tai dir doan, da bieu hien dirge protein hGM-CSF
trong chu chit to bao E. coil BL21(DE3)/pEThGM. De Idling djnh ket qua nay chi:mg tai bap
tut thvc hien phucmg phip Western Blot vai
2
'7
66

3

6
3
yaw

6

khang the (lac hieu khang hGM-CSF. Tren ban
phim xuat hien vach tin hieu tuong ang yen vi tri
cna each protein xuit hien trong phan doan chu
chAt khi duac cam frog IPTG (Hinh 4B, giLg 4),
ket qui nay khAng dinh vach protein dam xuat
hien khi duo: cam ang chink la GM-CSF. Tir
nhfIng ket qua ter'chung toi ket Juan da bieu hien
thanh ding protein hGM-CSF trong chu chit to
bao E. coil BL21(DE3)/pET-hGM.
Tinh such hGM-CSF trong chu chit to bio E.
coil BL21(DE3)/pET-hGM bing sic kI trao
di anion
Do protein hGM-CSF co pI 6 lchoang 5,5 la
mat protein china &lieu nham mang dien tich,
tich dien am trong dieu kien pH sinh ly (6-8). Vi
the chi:mg toi thvc hien khao sat tinh sach protein
nay bang sic kY trao dai anion, dung ly bing
each thay doi ming de) muSi hay pH vi cutii carig
danh gii mire da tinh sach.
7

1

2

3

4

5

6

7

ale

45
30

tale

20.1

14.4

le'

Ill

•

Hlnh 5. Kat qua tinh sach hGM-CSF bang phueng phap sac ky trao dai anion (cAt Q Sepharose FF), dung ly bang

tang nang d4 muai. A, SDS-PAGE; B, Western Blot vai khang the dac hieu khang hGM-CSF. 1. Thang protein
phan tCr luring thap; 2. Mau chu chat; 3. Phan Joan sau khi qua cat; 4. Phan doan tai can bang cat; 5. Phan doan rera
5 % ddB; 6. Phan doan dung ly 20 % ddB; 7. Phan doan dung ly 100 % ddB.
20,1 kDa cua thang phan tCr lucmg, tucmg Ong veri
KAt qua din di SDS-PAGE cho thay a cac
kich thirac dm protein hGM-CSF ly thuyet. 0 pH
gieng 2, 3, 4, 5 la min cac phan doan protein
dung djch can bang (pH,5) stn barn cna hGM-CSF
Wang ing vai phan doan protein tang sit, tit chu
vao chit trao dai anion 6 cat Q Sepharose FF gAn
chat, Olin doan protein sau khi qua cat, phan
nhu dien ra hoan town do khong co sv xuat hien
doan tai can bang cat va phan doan nfa, chnng raj
cda protein nay trong phan doan dung dich sau
titian thay 6 milli phan doan chu chat c6 su xuat
khi qua cat (Hinh 5A, gitng 3), mac du co sv raa
hien mat vach protein nAm gifta vach 14,4 kDa va
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Science & Technology Development, Vol 17, No.T4-2014
troi met phan hGM-CSF a giai down tai can bang
cat (Hinh 5A, gieng 4). d phan doan dung ly veri
20 % ddB, chimg toi they ce su xuAt hien met
vach protein dam, có kich thuac Wang Ung vai
kich thuac du doan cua hGM-CSF va toan be
protein do the duoc dung ly tai phan doan nay
(gieng 6) va khong can tai ruing de mad cao hon
(Hinh 5A, gieng 7). De khang dinh cac protein
tinh sach duoc chinh la hGM-CSF, chang toi tien

hanh lai voi khang the die hieu cho hGM-CSF.
Ket qua lai trinh bay trong Hinh 5B cho they cac
protein chitng toi thu duoc qua qua trinh tinh sach
bang sic ky trao dei anion chinh la protein mix
tieu hGM-CSF. Ngoai ra, mire do tinh sach cua
protein thu nhan duoc danh gin bang phan mem
xac dinh dam de va ket qua cho they de tinh sach
cua hGM-CSF thu duoc sau qua trinh sic kY trao
dei anion la 97,4 % (ket qua khong trinh bay).
Thir nghiem Wang ti,r Arai phucrng phap dung
ly bang pH, ket qua cho they bang each giam pH
kitting cho phep chimg toi thu duoc protein hGMCSF tinh sach (lc& qua khong trinh bay a day).
Nhu vay, tir nhftng ket qua ten chimg toi ket luAn

da tinh sach thanh ding protein hGM-CSF trong
chu chat to bao E. colt BL21(DE3)/pET-hGM
bang sac kV trao dei anion sir dung cot Q
Sepharose FF Arai phucmg phap dung ly tang
neng de mu6i.
KET LUAN
Nhu v4y, chUng toi da too clang thanh tong
Bong to bao E. coil BL21(DE3) co mang vector
tai to hop pET-hGM. DOng nay dr kha nang bleu
hien protein hGM-CSF trong chu chat to bao E.
coll. Su bieu hien hGM-CSF da throe kiem
chirng bang phuong phap SDS-PAGEva khang
dinh lei bang phucrng phap Western blot. Protein
nay duoc hen hinh tinh sach bang sac kV trao dei
anion sir dung cot Q Sepharose FF vai phircrng
phap dung ly tang ruing de muei. Mire da tinh

sach cila protein duoc xac dinh bang phan mem
dinh dam de voi ket qua dat 97,4 %. Chung nay
co the duoc sir dung lam ging de cung a/3
nguen nguyen lieu hGM-CSF cho quy trinh sin
xuAt hGM-CSF tai to hop ang dung trong y hoc
a Viet Nam.

Cloning, periplasmic expression and
purification of hGM-CSF (Human
Granulocyte - Macrophage ColonyStimulating Factor) in Escherichia coli
•
•
•

Phu Sang Nguyen
Thanh Thao Nguyen
Hieu Tran Van
University of Science, VNU-HCM

SUMMARY

•

Human GM-CSF is a cytokine consisting
of 127 amino acid residues, with four
cysteines being involved in two disulfide
bonds. Although GM-CSF is glycosylated in
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its natural form, the glycosylation perhaps

has not been involved in its biological
function. GM-CSF stimulates the survival,
proliferation, and differentiation of hem at


TAP CHI PHAT THIEM MN, TAP 17, Si 14-2014
transformants were induced by IPTG and
opoietic progenitor cells and also enhances
examined for hGM-CSF production.
the functional properties of mature myeloid
Periplasmic
proteins were released by
cells. GM-CSF is used as a therapeutic
osmotic shock treatment. The expression of
agent in various clinical cases such as
recombinant hGM-CSF was evaluated by
neutropenia following chemotherapy, bone
SDS—PAGE in total, cytoplasmic and
marrow transplantation, acute myeloid
periplasmic fractions. The recombinant hGMleukemia... In this study, we report the
CSF in periplasmic fraction was then
results on the cloning and expression of
subjected to ion exchange chromatography
recombinant human GM-CSF in the
using Q Sepharose FF column with saltperiplasmic space of Escherichia coll. The
increment elution step. SDS-PAGE showed
hGM-CSF gene was amplified by
there was a visible expression of
polymerase chain reaction using two
recombinant hGM-CSF in the periplasmic

oligonucleotide primers containing Ncol and
fraction of the E. coli BL21(DE3)/pET-hGM
Xhol restriction sites. This DNA fragment
and a purified band with the purity of 97.4%
was successfully cloned between the Ncol
after ion exchange chromatography. This
and Xho I sites of the plasmid pET-22b, in
result
was further confirmed by Western blot
frame with the pelB signal peptide sequence.
using anti-hGM-CSF antibody.
The expression vector pET-hGM was
transformed into E. coli BL21(DE3) and the
Keywords: hGM-CSF, periplamic, anion exchange chromatography.

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