Khảo sát sự biển hiện của GFP được dung hợp với HIS TAG tại đầu n hoặc c sử dụng plasmid pHT253 và pHT254 trong bacillus subtilis

TAP CHI PHAT TATA KAM TAP 17, SU 14-2014

Khao sat sty bi4u hiOn cua GFP duvc
dung hop voi HIS-TAG tai dau N hoc
C sir dung plasmid pHT253 va pHT254
trong Bacillus subtilis
•
•
•
•

Nguyin Hoai Nam
Phan Thi Phuvng Trang
Trin Linh ThuVc
Nguyin Dirc Hoang

Truirng D?i hoc Khoa hoc To, nhien, DHQG-HCM
( Ben nhOn ngay 15 thOng 09 min, 2014, nkin dang ngay 26 ththng 01 nam 2015)

TOM TAT
ma nguifri sO' dyng Ara chan chitin luqc dung
HO thong cac plasmid mang promoter
hop His-tag vao dhu N hohc C cOa protein
mpnh Pgrac100, pHT253 va pHT254 crow
sao cho hihu qua. Trong nghien cCru nay,
thuang mpi hem tren thi twang the gieri vao
GFP duqc chon nhu' met gen chi thi nhArn
nem 2012 da gbp phhn thOc day vi0c sir
khao sat anh btrang vi fri dung hqp cua Hisdyng Bacillus subtilis lam thong chti trong
tag len Al' bieu hihn trong B. subtilis. Ket qua
bleu hihn protein tai to hqp. Ngoai d6c dihm

cho they ss,r Neu hihn cua GFP trong 8.
mang mOt promoter manh, hai plasmid nay
subtilis giam rnpnh khi dung hop voi His-tag
can dugc tich hqp them duoi tinh the His-tag
tpi
dhu N. Day se la thong tin quan trong
trverc vOng MCS (Multi Cloning Site) trong
giOp Binh huong sO' dyng cOng nhu' thief ke
pHT253 (Pgrac100-8xHis-MCS) va sau wing
thi
nghihm trong bleu hihn tinh the protein
MCS trong pHT254 (Pgrac100-MCS-8xHis)
tai to hqp.
giap thuhn lqi cho viec tinh the va phat hihn
protein muc tieu. Thy terng protein muc tieu
Ter khda: Bacillus subtilis, GFP, His-tag, Pgrac100, pHT253, pHT254.

Mei DAU
Pgrac100 la met promoter manh cho Bacillus
subtilis, &roc dua vao sir clung clan tien trong

plasmid pHT100, da cho mac de bieu hien
protein muc tieu len den 30 % so ven protein tong
[1]. VOn la promoter the he thir 2, ke thaa nhang
tru diem cua promoter Pgrac [2]. Pgrac100 mang
nhang cai tien (i) yin-1g UP element giau AT, (ii)
vimg -35 mang trinh to bao heti TTGACA va (iii)
vung -15 mang trinh to ATG cho mac dO phien
ma tang gap 40 Ian [1] so veri Pgrac. Nhang ket
qua kha quan ve mac de bieu hien da cho thay

tiem nang to lem cua Pgrac100, gop met phan
khong nhet vao viec phat trien cua he thong bieu
hien pHT tr'en B. subtilis. Cimg vai nhang tru
diem On co nhu an town trong thixc phAm, len
men Cr mat de cao, kha nang het hieu qua [3], thi
viec sO hau met he thong bieu hien manh da giap
B. subtilis trer thanh met chisng this kha hty dan
trong linh vuc san xuat protein tai to hop.
Ben canh viec Iva chon mot he thOng bieu
hien phis hop, cho san luting protein cao thi hieu
qua tinh sach cang la mot nhu cAu quan trong
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Science & Technology Development, Vol 17, 1(1.14-2014

trong son suit protein tai to hop. Vai muc tieu
tao than Iii cho qua trinh tinh sach protein,
ngoai viec sir dung promoter manh Pgrac100, he
thong vector pHT con &roc dung hop them duel
tinh sach 8xHis-tag vao truetc va sau vung MCS,
tao ra hai phien ban pHT253 (Pgrac100-8.rHisMCS) N/A pHT254 (Pgrac100-MCS-8xHis) dtroc
phan ph& tren thi twang the gieri [4], cho phep
bieu hien protein muc tieu dtrai clang dung hop
8xHis-tag ,vao dau N hoac
C, theo thir to.
Van la met duoi tinh the phe bien, 8xHis-tag
mang nhieu tru diem nhtr kich thuac nha, ca the
dugc tinh the duoi dieu kien bien tinh, hien qua

tinh sach cao va
ding clang hoa [5]. Tuy
nhien, so bieu hien clia protein muc lieu co the se
bi inh hue/rig ben vi tri cua 8xHis-tag khi dung
hop vao dau N hoac C [6]. Do do, chang toi tien
hash kith() sit anh hoeing cua duei tinh the
8xHis-tag len so bleu 'hien ctla GFP (Green
Flourescent Protein) khi dung hop vac) du N
ho5c C trong B. subtilis, trong twang hop nay
GFP nhtr met protein chi thi. Anh huemg cua
du& tinh the His-tag len so bieu hien protein
muc tieu se dupe danh gia doa vao ket qua phan
tich SDS-PAGE vi kha rang Oat huynh quang
trong mei truemg ran vi long tren B. subtilis.

a

VAT LIEU VA PHUONG PHAP
Chiing vi sinh vet, plasmid vi mai trwang nuoi
A
cay
E. colt OmniMAX (Invitrogen) dune sir dung
6 bulk dong hoa va B. subtilis 1012 &roc ding
de danh gia inh htremg cua 8xHis-tag len so bieu
hien cita GFP trong thi nghiem nay. Ccic plasmid
dune sir dung bao gem pHT254 cilia promoter
Pgrac100 vi gen 8xHis am sau vung MCS;
plasmid pHT1169 di dtroc tao ra trong met de
tat khac bang each chen gen chi thi gfp vao Yung
MCS cua plasmid pHT253 (CO eau trite tuong or

pHT254 nhung gen 8xHis nam truac Yung MCS).
Te bao B. subtilis dune nal cay lac tren mei
true:mg Luria broth (LB) a 37°C, vai khing sinh
chloramphenicol 10 Itg/mL.
Trang 6

Thiet ke plasmid pHT1170
Doan gen gfp dtroc thu nhan bang kT that
PCR Yeti khuon la pHT1Otgfp.[3] va cap mei
ON1277(5'AAAGGAGGAAGGATCCATGGCT
AGCAAAGGAGAAGAACT-3'),ON1278(5'GGCCATGACGTCTTTGTAAAGCTCATCCAT
GCCATGTGT-3'). San pham PCR dupe x.r ly Yeti 2
enzyme BrunH1 va And' va not vao pHT254 cling da
dupe xu ly cling loai enzyme tao thanh vector
pHT1170 (Hinh 1B). Qua trinh sang loc dupe thoc
hien trong E. colt Omni MAX vdi mei ON1249(5'CGTTTCCACCGGAATTAGCTTG-3'), ONI359
(5'ACGTACGATCTTTCAGCCGACTC-3'),
plasmid dung dupe bien nap vao B. subtilis 1012.
Khali) sit hoot tinh GFP tren mai trirang rife
Cac khan lac don B. subtilis china plasmid
pHT I 169, pHT1170 dupe khao sat kite rang bieu
hien GFP nham danh gia anh huang cda 8xHistag. Chain cac khan lac B. subtilis len dia LBAgar-Cm yeti 6 ruing de IPTG (0 mM; 0,001
rnM, 0,01 mM; 0,1 mM; 0,5 mM va 1 mM). U a
37°C trong 16 gia. Sr phat huynh quang cda
khan lac dtroc kich thich dual budtc song UV,
mire do' pith huynh quang phan anh mac de bieu
hien cda GFP, qua do danh gia dune mire de bleu
hien cita GFP tai to hop vii His-tag. Hinh inh
dupe ghi nhan bang may chop hinh ks, that se vi
dune phan tich bang phtm mem AlphaEaseFC

(Alpha Inotech) de tinh tan gray value (hay
pixel value). Thoc hien Wang to vii chimg am lit
pHT01. Thi nghiem dune lap lai 3 lan, m8i Ian
tren 3 khuan lac khac nhau.
Cam sing bieu hien GFP
Ching B. subtilis 1012 china cac plasmid
pHTI169, pHT1170 dune nal cay trong mei
tn.remg LB chira khang sinh chloramphenicol (10
1.T/flip a 37°C va tOc d lac 250 vong/philt. Cam
(mg bang IPTG tai thati diem ODA dat 0,8 — 1
vii cac ming de 0 mM; 0,001 mM; 0,01 mM; 0,1
mM; 0,5 mM vi 1 mM. Thu mau 6 0, 2, 4 gi6 sau
cam ling. ly tam 6000 yang/phut trong 3 phitt de


TAP CB PRAT TRIfIl KUM TAP 11, Si T4-2014
thu sinh Ichei va gift mitt a -20°C. Tien hanh
tucrng to va thu man a thai diem 4 gier sau cam
ung vai mau chimp pHT01. Thi nghiem &roc lap
lai 3 lan tren 3 khan lac khac nhau. Mau sau khi
thu se &lac ding de dien di SDS-PAGE vi !dial)
sat hoot tinh GFP tren dia 384 gieng.
Kant tra ay bieu hien bing SDS PAGE
I-16a tan sinh khei trong 60 ul PBS (Phosphate
Buffer Saline), them lysozyme dot neng de cuei la I
ug/p.1, 6 6 37°C trong 5 phfit, sau do them 30 id
loading buffer 5X (0,135 M Tris/HC1, 30%
glycerol, 3% SDS, 0,03% bromophenol blue, 0,15
M DTT). Mau sau khi ly se &cm u 6 95°C trong
5 phit, ly tam 10.000 vang/phtit trong 5 phfit vi thu

dich not de tien hanh din di SDS-PAGE. Dinh gia
anh hueng dm His-tag tai dau N vi deu C len so
bieu hien cua GFP.
'Chao mit haat tinh GFP tren dia 384 gieng
Kha tang phat quang c6a GFP lien quan tai
met du trim Chromophore, duos tao thanh bed 3
acid amin Serine65-Tyrosine66-Glycine67 tai
trong tam host Ong dm churl polypeptide. Qua
trinh ding yang la met chwii cac phan img khi
hydro vi oxy hoi, tao ra plan to Chromophore
trueng thinh cho phep hap thu sinh sing toi da
btrac sting 475nm vi phat huynh quang mau xanh
loc ce buck sang 508 nm [7]. Mau to bio B.
subtilis 1012 mang cite plasmid can kheto sit

duac thu a buck cam img bieu hien se &roc phi
bang dung dish PBS 1X (Phosphate Buffer
Saline) yeti a:mg de lysozyme 10 og/ul, fl IL
trong 30 plait. Dich chiet to bao sau ly tam &Km
do tren dia 384 gieng vii may doe dia (BioTekThermo, My). Ket qua dtroc xu ly vi tinh town
bang Olen mem Excel (Microsoft). Thi nghiem
duoc lap lai 3 lan tren 3 khan lac khac nhau.
KET QUA.
Da dimh gia inh hiring dm His-tag len khi

nang bleu hien GFP, chung t8i thiet ke plasmid
pHT1170 &Km dieu hos phien ma bed promotor
Pgrac100, cho phip Neu hien protein GFP dung
hop vii 8xHis-tag tai dau C cfia protein vii cic
phucmg phip da dirge de cap 0 phan Vat lieu

phuang phip. Trong nghien cfru nay, plasmid
pHT1169 (Hinh 1A) diroc si chmg thu met del
cluing do cO cau trite tucmg to pHT1170 (Hinh
1B), nhung 8xHis7tag se chrac gan vat) dau N ctla
GFP 6 clang dung hop. Cie cheng B. subtilis
1012/pHT1169 (Pgrac100-8xHis-gfp) vi B.
subtilis 1012/pHT1170 (Pgrac100-gM-83cHis) se
duac gift chfmg vi su dong cho cac thi nghiem
khao sit 6 phin ;au. Bing each so sinh hai clang
dung hop 8xHis-GFP va GFP-8xHis, cluing WU se
danh gia &roc inh hiring dm His-tag len gr bleu
hien vi phat huynh quang cua GFP.

B

A

Ami_

picritro

pHT1169
ORF-.2
1

e6S bpe

ORF-2

$142 bps

Pgravt“
fix1411,ptp

ORF-3

40°

Vprac100
gip

!babe
OAF.

RPM
BOON

OAF.

40"

Na and
• /huh I. Sa de plasmid pHT1169 (A) va pHTI170 (B), vat vi tri gen 8xliis a trmic va sau gene', too 2 dang dung
hop 8xHis-GFP va GFP-8xHis theo thin to.

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Science 8 Technology Development, Vol 17, No.T4-2014

Danh gia anh huiing caa SxHis-tag khi nuoi
cay cluing tren moi trvong ran
Kha nAng bieu hien GFP cua hai chting B.
subtilis 1012 mang plasmid pHi 169 va pHT1170
chrqc quan sat thong qua kha rang' phat huynh
quang khi chArn khutin lac tren cac &a thach chaa
khang sinh va 6 nong de IPTG khac nhau. Ket
qua sau khi a 16 gia qua dem 6 37 °C dtrqc ghi
'than nhu a Hinh 2A. Gia trj cuerng de sang ctia
khutm lac cluqc tinh town bang phan mem
AlphaEase, qua do phin anh mac de bieu hien
GFP
2B).

A

Quan sat ket qua Hinh 2, cluing tot !than thAy
de sing dm pHT1169 so veri chimg am pHT01
thi khong co sir khac biet nhieu o tat ca cac nong
de IPTG. Tronglchi do, pHT1170 cho kha nAng
phat huynh quang manh vi tang clan theo nong de
chat cam (mg, dat etre dai ar ',hang nong dt cao
(0,1; 0,5 vi 1 mM IPTG). Mac de bieu hien thap
cua chin mang plasmid pHT1169 so vii
pHT1170 cho thAy His-tag co anh huang den
protein muc tieu, cu the la viec dung hop vao dAu
N lam giant sir bieu hien cua GFP. Ket qua nay se
&roc kiem chain bang cac tht nghiem tiep theo.

B. subtilis 1012

Gray value

0 0,001 0,01 0,1 0,5 1 mM

pHT01

IPTG

B

120
100
80

pHT1169

60
40

pHT1170

20
0

pHT01 pHT1169 pHT1170
Huth 2. Ket c ua phat huynh quang GFP tren dTa thach chira IPTG (A) va pilau tich de dam bang AlphaEase (Alpha
Inotech) tai nong de 0,1 mM IPTG (B) cila B. subtilis 1012 mang vector pHT1169, pHT1170, sir dung chimg Am hi
pHT01.
Danh gil anh htrang curt 8xHis-tag trong mai

truing long sir dung SDS-PAGE

Hai cluing B. subtilis 1012/pHT1169 va B.
subtilis 1012/pHTI 170 cluqc nudi cAy cam Ong
cling 1 dieu kien nhu dA me too phan Vat lieu
phtrong phap. Mau duqc thu a cac th6i diem 0, 2,
4 gier sau do ly tam thu sinh khOi, phi Ink va
tiAn hanh dien di SDS-PAGE. GFP co kich thuac
Wong ducmg la 27 kDa, gAn vach 30 kDa cua
thang chuttn. Ket qua bleu hien cua GFP dung
hop sten His-tag 6 dAu N va C dime the hien a
Hinh 3. So skill vii ching Am pHT01, chin B.
sztbtilislpHT1170 (Hinh 3B) cho kha nAng bieu
hien GFP virgt mix 6 nhang nong de) IPTG cao

Trang 8

(0,1, 0,5 WI 1 mM) Ira tang dtin theo th&i gian
cam (mg. Tuy nhicn, vach protein muc tieu nay
lai rat kho nhan thAy del vii chung B.
subtilis/pHT1169 (Hinh 3B). Nhu vay, mac de
bieu hien cda GFP dirge dung hqp 8xHis-tag
dau N (pHT1169) giam han rat nhieu so v6i dang
dung hop 6 dau C (pHT1170). Dieu nay hoax'
toan trong khop vii thi nghiem khito sat hoat tinh
tren &a thach. Su anh huang nay se duqc }dem
chimg met lin naa bang plan:mg phap khao sit
cuing de phat huynh quang tren &a 384 gieng.

TAP CHI PHAT HIEN
ao

Iv&

TAP 17, Si 142014

nang bieu hien tang din theo nOng de IPTG va
thai gian cam img, dat cue dai tai 0,5 mM IPTG
sau 4 gia .cam img. Doi yen pHT1169, muc do
bleu hien kha thap clan den su che'nh lech giUa
cac nghiem thirc la khong ding ke. Ket qua nay
met Fan naa khiing dinh vies His-tag dung hop
vai protein GFP tai dAu N da anh haling den su
bieu hien ctia protein nay trong B. subtilis. Trong
khi, His-tag a dttu C cho mire de bleu hien kha
tot, cao gAp nhieu ltin so vai twang hop con 14i.
Thio luin

Huth 3. Kat qua khito sat Mau hien cita GFP bang
phuong phap dien di SDS-PAGE 2 chang B.
subtilislpHT1169 (A) va B. subtilis/pHT1170 (B). M,
Thang protein; (-), pHT01. MAu thu 60, 2, 4 gia sau
khi cam img vai IPTG b cac 6 nAng do khac nhau.
Dinh gii anh hiring elm 8xHis-tag trong mai
truing long sir dyng phirong phip do host
tinh teen dia 38-1 gieng
Phuang phap do cueing de huynh quang co de
chinh xac cao han 2 phixang phap tren. Wing de
GFP cang cao thi gia tri do huynh quang cang 16n

va nguoc lai, qua do c6 the dinh gia mile dO bieu
hien ciut GFP nhim dua ra &we cac thong se
khach quan ve anh huong cita duoi dung hop
8xHis-tag. SO lieu ductc xir ly va trinh bay dual
Bang de thi Hinh 4. So sanh ctrang de phat huynh
quang cint 2 chung mang plasmid can khao sat,
chung toi nhan thtty pHT 1170 (-1600) cho kha
nang bieu hien GFP cao Op 20 lin so vai
pHT1169 (-80). Trong do, pHT1170 cho kha

De co the phien ma, RNA polymerase se phai
barn vao promoter va trot, viec xuat hien met
doan gen 8xHis nim truac gen muc tieu c6 kha
nang se anh hulling den qua trinh phien ma. )(et a
mire de dich ma, de co the tong hop duoi His-tag,
to bao phai huy dung cilng luc nhieu histidine,
dieu nay se gay met su Un tic, lam cbAm qua
Wirth dich ma neu nhu tinh trang thieu but t-RNA
mang histidine dien ra. Hoc ea kha nang, dau N
cita GFP c6 vai trO quan trong trong viec bieu
hien cling nhu quyet dinh hoat tinh sau khi dich
N se anh
ma, duel poly-histidine xuat hien 6
huerng sden qua trinh gap cuOn, lam giam hog
tinh ciia protein nay [6].
Trong met nghien cim khac (ket qua chtra
ding be), nhom chimg t8i cang da khao sat anh
huang clia His-tag len si,r bieu hen cita
galatosidase (BgaB), kha rang bieu hien dm
BgaB khi dung hop vai His-tag a &Au N giam

hon 3/4 so voi dAu C. Nhang ket qua c6 duoc holm
toan phi hap gift hai nghien ciru, viec dung hop
His-tag vac) dAu N da anh huang rat lon den si,r
bieu hien ciia 2 protein chi thi GFP va BgaB.
Nhung day khong phai la thOng tin chung ma tity
thuec vao protein muc lieu.

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Science & Technology Development, Vol 17, No.T4-2014
B. subtilis

1012 / pHT1169

Don vj hoat tinh

1012 / pHT1170

2000

• 0 mM

80

B. subtilis
Dom vi hot tinh

M0,001

1B00

• 0,01

1 200

• 0,1

40

■ 0,5

BOO

• 1 mM

400
0 gib

2 gib

4 Ws

0 o gib

2 gib

4 gib ThM

1-finh 4. Ket qua khao sat hoot tinh GFP B. subtilis 1012/ pHT1169 (8xHis-GFP), B. subtilis 1012/ pHT1170 (GFP8xHis).

KET LUAN
Tir tat ca cac ket qua dat dirge, chnng toi co
nhffng nhan xet nhu sau: kha nang bieu hien GFP
cua plasmid pHT1169 giam manh khi then them
gen 8xHis vao trunk gen gjp; trong khi do, dOi
yeti plasmid pHT1170 (cling chira promoter
Pgrac100, dudi His va gen gfp), nhung luc nay
gen 8xHis /lam sau gen muc lieu thi kha nang
bieu hien GFP cao hon gap nhieu lam Dieu nay

cho May vj tri dung hop cila His-tag er dau N
lam giam bieu hien so v6i Tau C vai chi thi la
GFP. Day cling se la mOt thong tin quan trong
trong chien ltroc sir dung pHT253 va pHT254.
Nghien ctru nay &tor tat tro bed 121.12 phat tplin khoa
hoc va tong nghe au& gia (NAFOSTED) trong de tai
and so 106.16-2011.80.

Investigating the expression of GFP
fused with HIS-TAG at N- or C-terminus
using plasmid pHT253 and pHT254 in
Bacillus subtilis
•
•
•
•

Nguyen Hoai Nam
Phan Thi Phuong Trang
Tran Linh Thuoc

Nguyen Duc Hoang
University of Science, VNU-HCM

ABSTRACT
pHT253 and pHT254 belong to the
plasmid system carrying Pgrac100, a strong

the use of Bacillus subtilis as host for the
production of recombinant proteins. Along

promoter, which were commercialized in the

with strong promoter Pgrac100, these two

global market in 2012 have pushed forward

plasmids were added His-tag to before and

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TAP CHI PHAT TRIEN HUN, TAP 17, SO T4-2014
after MCS (Multi Cloning Site) in pHT253
(Pgrac100-8xHis-MCS) and pHT254
(Pgrac100- MCS-8xHis), respectively, to
facilitate purification steps. Depending on the
farget protein, the user selects appropriate
effective strategy of fusing His-tag, i.e. either
into the N- or the C-temimal of protein. In this
study, the GFP gene was used as an

indicator gene to investigate the influence of
the His-tag position on the protein
expression in B. subtilis. Our results showed
that the expression of GFP in B. subtilis
significantly was reduced in the fusion form
with His-tag at the N-terminal. This would be
important information for the selection of
suitable vectors.

Key words: Bacillus subtilis, His-tag, GFP, Pgrac100, pHT253, pHT254.

TAI LIEU THAM KHAO
[1]. T.T.P. Phan, H.D. Nguyen, W. Schumann,
Development of a strong intracellular
expression system for Bacillus subtilis by
optimizing promoter elements. Journal of
biotechnology, 157, 1, 167-172 (2012).
[2]. H.D Nguyen, T.T.P. Phan, W. Schumann,
Expression vectors for the rapid purification
of recombinant proteins in Bacillus subtilis.
Current microbiology, 55, 2, 89-93, (2007).
[3] S.L. Wong, Advances in the use of Bacillus
subtilis for the expression and secretion of
heterologous proteins. Current Opinion in
Biotechnology, 6, 5, 517-522 (1995).
[4] Bacillus subtilis Pgrac100 Expression
Vectors: />ucts/bio/04_vector_sys/b_subtilis_pgrac100
.html
[5] E. Hochuli, W. Bannwarth, H. DObeli, R

Gentz, D. Saltier, Genetic approach to

facilitate purification of recombinant
proteins with a novel metal chelate
adsorbent. Nature Biotechnology 6, 11,
1321-325, (1988).
[6] P. Ledent, C. Duez, M. Vanhove, A.
Lejeune, E. Fonze, P. Charlier, F. RhaziFilali, I. Thamm, G. Guillaume, B. Samvn,
B. Devreese, J. Beeumen, J. LamotteBrasseur, J.M. Frere, Unexpected influence
of - a C-terminal-fused his-tag on the
processing of an enzyme and on the kinetic
and folding parameters. FEBS letters, 413,
2, 194-6(1997)
M.
Ormo, A.B. Cubitt, K. Kallio, L.A
[7]
Gross., R.Y. Tsien, S.J. Remington, Crystal
structure of the aequorea victoria green
fluorescent protein. Science, New York,
KY., 273 ,5280, 1392-1395 (1996).

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