Cấu trúc chủng nấm men pichia pastoris biểu hiện mini proinsulin dạng tiết ra môi trường nuôi cấy

TAP CHI PHAT TRIA KH&CN, TAP 15, SO T1- 2012
CAU TRUC CHUNG NAM MEN PICHIA PASTORIS BIEU HIEN MINI-PROINSULIN
DANG TIET RA MO! TRUtiNG NUOI CAY
Ngo Thi Kim Hang, VO Minh Tri, Tran Linh Think

Trtrang Dai hoc Khoa hoc Ttr nhien, DHQG-HCM
(Bai &kin nay 21 thcing 03 nam 2011. holm chink stia chat, ngay 09 thcing 04 nom 2012)

TOM TAT: Dai thao dwang la mat trong nhiing can benh clang nit chivy span tam hien nay.
Nham muc click san xuat insulin tai to hgp phyc vu (Reit, trj benh clai thao &rang, chang toi clang 126a va
biau hien miniproinsulin trong Pichia pastoris. Doan DNA mang gen ma h6a miniproinsulin-6xHis
(MPI-6xHis) cluvc dung haa vao vector pPICZaA too plasmid pPICZaA/h-mpi de: cdu trzic cluing P.
pastoris GSI 15::h-mpi bidu hien MPI tai to hgp clang tilt ra ngoai mai truang. Kidzi hinh va kieru gen
czia P. pastoris GS115::h-mpi clucrc xac djnh bang PCR va cat hgn che. MPI clugy bizt hien a clang
dung hgp vai th'd 6xHis khi cam zing vol methanol vai ming clO cuai cling 0,5% sau 96 gier.
Tir khda: biju hien gen, Pichia pastoris, mini-proinsulin
MD DAu

tinh. Gan day, mO hinh mini-proinsulin (MPI)

Dai thao dutmg (DTD) la mOt trong nhimg

da &roc nghien cau vai nhieu uu diem. Mini-

can benh dang duce quan tam hang du hien

proinsulin c6 eau trizc tuang ttr vai proinsulin,

nay. Benh DTD, chitin khoing 60-70% trong

chi khac la c6 cai tien thay the doan peptide

cac benh nOi tier, la nguyen nhan gay tir vong

du carboxyl (dau C) tu nhien 31 amino acid

dimg hang thin nam 6 cac mac Oat trien va

trong proinsulin bang mOt doan peptide ngan

dang tang dOt bien a nhieu nuac dang phat

hon (9 amino acid), giup di clang cho viec tinh

teen,' cac nu& ding nghiep mai. Hang nam

che va gia tang hieu qua Op cuOn dAn den tang

tren the giai c6 khoang 3,2 trieu ngutri chez vi

hieu suet hinh thanh insulin c6 hoat tinh hcm

benh DID [4]. Ngay nay, viec sir dung insulin

mo hinh proinsulin. Doan peptide C ngan han

ducrc xem IA mOt phan kh6ng the thieu doi vai

lam tang kha nang gAp cuOn len 20-40% so vai

benh nhan mac benh DID, dOc biet la DTD tjrp

proinsulin 6 citing n6ng d6 [1].

1 [6]. VI the, viec san xuat insulin ngutri bang

Pichia pastoris la he thong bleu hien protein

con &rang protein tai to hap da va dang la giai

ngoai lai dang ducrc cac nha khoa hoc chit 5, bai

phap hieu qua nhAt hien nay.

nhUng dac tinh uu vi'et nhu co the thtrc hien

Phtrcmg phap san xuat insulin ngoai tai to

rthung bien dOi sau djch ma, nuoi coy a mat d6

hop dtra ten mOt chuai protein (proinsulin)

cao to hrong sinh khOi lan trong qua trinh len

thay vi 2 chuOi da cluvc khang djnh uu the ve

men, thao tac de clang [3]. Ngoii ra, he thong

Icy thuAt va bleu qua thu nhAn insulin c6 hoot

P.

pastoris co the san xuat Itrong 16n protein
Trang 45


Science & Technology Development, Vol 15, No.T1- 2012
muc tieu vai nguAn nguyen lieu methanol re

moi 5'AOXI va 3'AOXI (Invitrogen) duce

tien va it bi tap nhiem.

dung di sang iQC va kiem tra plasmid tai to

N1Itn muc dich san xuat m'Ot hum km
insulin tai t6 hqp cua nguei de phyc vu diEu tri
benh dai than dueng, chimg t8i tiEn hanh

cau

true chang nam men P. pastoris GS1I5::h-mpi

hqp, giai trinh to d6ng thei kiem tra kieu gen
the bien nap P. pastoris.
Cu trac chung E. coli 0115a mang plasmid
tai to hqp pPICZaA/h-mpi

bieu hien MPI tai to hqp clang tiet vao trong

Quy trinh tao clang E. coli DH5a mang

moi tndyng nuoi cay. Klett hinh va kieu gen cCia

plasmid tai to hop pPICZaA/h-mpi dugc thuc

chang P. pastoris GS115::h-mpi ding nhtr kha

hien nhu sau: thu !than gen h-mpi ma Ma

nang bitu hien protein dung hop MPI-6xHis

protein mini-proinsulin dung hqp vai the 6x

dang tier duqc xac dinh.

histidine (6xHis-MPI) bang PCR to khu8n la

VAT

Lieu VA PHYONG PHAP

plasmid pMZZIn sir dung cop moi dac bleu
5 'PHTI Xho va 3 'PHTI Xba. Chuang trinh

Chung vi sinh vet, plasmid va moi
Chang chit E. coil DH5a [F, 080IacZAM15,

chay PCR: 95 C/5'; 30 chu ky: 94 C/1',

recA 1, endA1, hsdR I 7(rk-, mk+), phoA, supE44,

55 C/45", 72 C/2'; kEt th6c: 72 C/10'. San

thi-1, gyrA96, relAlj duqc dung dE dong

pham PCR duce cat vai Xhol va Xbal de tao

hoa

cac gen (Takara). Chang Pichia pastoris

dau

dinh; that hien phan Ong not gen h-mpi

GS115 [Mutt, his4] duqc sir dung de bleu hien

vao pPICZaA da duqc cat ma yang bang

protein muc tieu.

enzyme ttrcmg img de hinh thanh plasmid tai to

Plasmid pMZZIn CO ngu6n g6'c to plasmid
pUC 19 mang doan gen h-mpi dtrqc dung

lam

khuon trong PCR thu nh(in gen h-mpi.
pPICZaA (Invitrogen) la vector sat nhap vao P.

pastoris c6 kich thuac khoing 3,6 kb, mang
gen khang Zeocin, trinh tu tiet a-factor cho
phep bleu hien protein muc tieu a clang

hqp pPICZaA/h-mpi. pPICZaA/h-mpi dtrqc
bin nap vao E. coli DI-15a bang phtrang phap
Wen nap calcium lanh [8]. Deng to bao E. colt
DH5a mang pPICZaA/h-mpi duqc sang 19c
tren moi trutmg LB agar chaa Zeocin n6ng d6
cu6i la 25 pg/m1 va kiem tra deng muc tieu
bang PCR khuan lac vai cap moi 5'AOXI va

Gen mix tieu dtrqc diEu khien bai promoter

3'AOXI. Chuang trinh chay PCR: 95 C/5'; 30

5 'AOX1 .

chu ky (94 C/1', 55 C/45", 72 C/2'); 72 C/ l0'.

Cap moi 3 'PHTI Xba (GTA CTC TAG ATT
AGT TAC AGT AGT TCT CCA G) va
5 'PHT1 Xho (ATG CCT CGA GAA AAG
AGA AGA AGC TGA AGC TGA AGC TCC
AAA GCA TCA CCA TCA CCA TCA CGG
TGG AAG ATT TGT CAA CCA ACA TCT
GTG) duqc dung de khuech dal gen h-mpi. Cap
Trang 46

Deng to bao mang pPICZaA/h-mpi cho san

pham PCR vai kich thtrac 751 by dtrqc kiem
tra ten gel 1% agarose. Cac th'E Win nap duqc
tach chiet plasmid bang phixong phap SDS
kiem [8]. Plasmid sau khi thu nhan duqc kiem
tra bang PCR plasmid (sir dung cop mil
5'AOXI va 3'AOXI) va phircmg phap cat hp


TAP CHI PHAT TRIEN KH&CN, TAP 15, S6 Tl- 2012
cite vai Xhol va Xbal. Nhang plasmid duang

methanol vao moi truang BMMY nu6i cAy de

tinh dugc giai trinh to vai kit BigDyeTM

ctat n6ng rt6 at& 0,5%. Sau 96 gib thu dich moi

Terminator (Macrogen Inc., Han Quec). Ket

throng, b6 sung PMSF (phenyl methyl

qua giai trinh to dugc so sanh di) tucmg diing

sulphonyl fluoride) de dat ang dQ cu6i la 1

vai trinh tir 19 thuyet bang pilaf.' mem Jellyfish.

mM va 6 -30 C.

CAu trot cluing P. pastoris GS115::h-mpi

mang plasm id tai to hqp pPICZaAnt-mpi

Su bleu hien protein muc tieu MPI-6xHis
dugc kiem tra bang SDS-PAGE va khing dinh

Nham tang hieu suit thanh ding trong qua

lai bang lai Western sir dung khang the dac

trinh sat nhap plasmid vao b0 gen P. pastoris,

hieu khang insulin HU1018 (dugc cung cap tir

pP1CZaA/h-mpi dugc xir 19 veil Sad thanh

tien sy Peer Nobert Jorgensen (Novo Nordisk,

dung thing. Sau dO, plasmid nay dugc bien nap

Dan Mgch)) va phut hien nh6 khang the anti-

vao P. pastoris GSI 15 bang xung dien vai hieu

mouse IgG-HRP (Abcam).

dien the 2,5 kV, dien tra 200 1-2 va thoi gian

KET QUA VA THAO LUhd•I

phut sung la 5 mill giay. Dong to bao P.

Ciu true chting E. coil DH5a mang

pastoris tai to hgp dugc sang I9c tren mei

pPICZaA/h-mpi

a dat dmg

Plasmid pMZZIn mang gen h-mpi dugc clUng

dp cti6i 100 gg/ml. Sau do cac the bi&I nap

lam Ichuon trong PCR thu nhan gen h-mpi bang

dugc kiem tra kieu hinh dua tren su ph& triAn

cap m6i 5 'PHTI Xho va 3 'PHTI Xba. San

diing deu tren moi tru6ng MDH (minimal

pham PCR cho kich thuac khoang 250 by phit

dextrose with histidine) va MMH (minimal

Nip voi kich thtrac du kien la 263 by (Hinh 1,

methanol with histidine); kiem tra kieu gen

gieng 2). Cac the bien nap dugc sang I9c bing

bang PCR voi cap moi 5'AOX1 va 3'AOX1.

PCR khuin lac vai cap moi 5'AOX1 va

Chuang trinh chay tucrng tke nhu PCR khuin

3'AOX1. Plasmid tiz nhang the Wen nap cho

lac.

ket qua PCR khuin lac &rang tinh dugc tach

Xac dinh kha nang bleu hien MPI clang tiet

chi& de kiem tra bang PCR va cit hart the

ra ngoai mot trtrimg nuoi ciy

(Hinh 2). Su hien dien cua gen h-mpi dugc

tnrong YPDS agar b6 sung Zeocin

Cam ang bieu hien P. pastoris GS115::h-mpi

kiem tra bang PCR vai cac cap m6i 5'AOX1

bang methanol: CAy mot khuin lac don vao 50

va 3'AOX1 (bit cap dac hieu ten vector) va

ml BMGY va nuoi ciy lac 6 30 C qua dem cho

5'PHTI-Xho va 3'PHTI-Xba (bit Op dec hi0

den khi OD580 dat 2-6. Ly tam 4000 rpm, 5

ten gen h-mpi).

phut a 4 C. Chuyen sang 200 ml BMMY de
cam Ong su bieu hien protein num tieu (0D580
= 1 a thai diem 0 h). Sau moi 24 gib b6 sung

Trang 47


Science & Technology Development, Vol 15, No.T1- 2012
2
6000 bp -

.111•11■

30(H) bp-

■

1111••••

6000
4000

3000

,-.. NI hp I 000 by

by

bp

3505

2000
1500

-

1000

!WO by VW by -

3755

751

7541

163 hp

589

500

263

250

Hinh 1. Dien di do san phim PCR gen h-mpi tir
plasmid pMZZIn (2), thang chair DNA 1 kb (1).

Hinh 2. Dien di do san phim PCR cila pPICZaA (2),
pPICZaA/h-napi bing cap moi 5 'AOX1_3'AOX1 (3),
pPICZaA/h-mpi bAng cAp moi 5 'PHTI-Xtio va 3'PHTIXba (4), San phAm cat pPICZaA bAng XhoI (5), San phim
pPICZaA/h-mpi bAng XhoI (6), pPICZaA/h-mpi bang
XhoI va XbaI (7), that's; did') DNA 1 kb (1).

San pham PCR cua pPICZaA/h-mpi (Hinh 2,
gieng 4) co kich thuac khoang 250 bp, tucmg

CAti true P. pastoris GSI15::h-mpi mang
pPICZaA/h-mpi sat nh4p vao V) gen

trng vOi kich thuorc gen h-mpi &rot khuech dai

pPICZaA thu6c dang vector sat nhap, khi

bang moi dac hieu 5'PHTI-Xho va 3'PHTI-Xba

bien nap n6 sat nh'ap vao b6 gen cua to bao chit

(263 bp). Ben canh dO, san phA'm PCR plasmid

th6ng qua tai to h9p tucrng d6ng. pPICZaA/h-

tai to hop vai op moi 5.AOX1 va 3'AOXI CO

mpi dupe bien nap vao P. pastoris GS115 bang

kich thuerc khoang 751 by tucmg Ung vai gen

dien Wen nap. Cac the bien nap duqc sang 19c

h-mpi (263 bp) cong 1 phan trinh to giaa hai

dua tren kha nang khang khang sink zeocin.

moi tren vector (Hinh 2, gieng 3). San pham

Nhimg khuan lac du tuyen dtrgc thu nhan va tiep

pPICZaA/h-mpi cat bang XhoI va Xbal cho hai

tuc kiem tra kieu gen va kieu hinh sir dung

vach tucmg dng vai kich thuac pPICZaA (3505

methanol.

bp) va gen h-mpi (263 bp) (Hinh 2, gieng 7).
Trong khi d6, san pham PCR pPICZaA (khong
mang gen h-mpi) bang

op moi 5'AOX I va

3'AOX I c6 kich think 589 by (Hinh 2, gieng
2).
pPICZaA/h-mpi chi a gen muc tieu h-mpi

Kieu hinh va kieu gen cac P. pastoris
GS' 15::h-tapi
Sau khi sang 19c tren moi truCmg YPDSZeo100, cac the bien nap (trot kiem tra kieu
hinh bang each ria d6ng thCri len moi trutmg
MDH va MMH. Dip vao

tic do phat trien cda

(lucre giai trinh to de kiem tra c10 chinh xac ve

chi:mg tren ca hai moi truong de xac dinh kieu

trinh to cang nhu da d6ng khung dich ma.

hinh (Hinh 3).

Trinh to nh:an clugc c6 dcj tucrng d6ng 100% va

dOng khung clich ma vai trinh to ly thuyet.

Trang 48


TAP CHI PRAT TRIOI KUM, TAP 15, SO T1- 2012

Hinh 3. Kieu hinh the hien nap GS115::h-mpi ten mbi trireng MDH (A) va MMH (B).
MDH la mei trutmg t6i thieu china

khong co. Deng thtri d6i vai cac chang mang

dextrose con MMH IA mai throng china

gen h-mpi xuat hien vach DNA c6 kich thtrac

methanol lam ngutin carbon duy nhAt. The bien

751 by (Hinh 4, gitng 4)

nap dugc trai tren hai mei trtrOng, neu t6c d6

Cac the bien nap c6 kieu hinh Mut+ cho

tang tracing deng deu thi chting to the bien nap

hai san phial PCR: 751 by (do cap mei bat cap

c6 kha nang sir dung methanol mph hay not

vai trinh to arcmg deng 5'AOXI va 3'AOXI

cach khac, chnng c6 kieu hinh Mut+ (Methanol

tren plasmid pPICZa/h-mpi da sat nhap vao be

utilization plus). Ngtrqc lai, neu the bien nap

gen) va 2200 by (do op m6i bat cap v6i trinh

tang trutmg cham tren mei twang MMH thi se

ttr 5'AOX1 va 3'AOXI sin c6 tren be gen dm

c6 kieu hinh Mut'. Qua lc& qua tang truOng

P. pastoris GS 115) (Hinh 4, gieng 4). Trong

tren hai mei trutmg MDH va MMH, cho thay

kfii de, be gen cua P. pastoris kitting dugc sat

cac the bien nap chqn lqc dugc c6 kieu hinh

nhap bed pPICZa/h-mpi cho met vach 2200 by

Mut'.

do m6i chi bat cap vai trinh ttr 5'AOXI va

De xac nhan su phu hqp gift kieu hinh va

kieu gen mang gen h-mpi, DNA b6 gen dugc ly
trinh tir cac the bien nap va thuc hien PCR bang

3'AOXI san c6 tren be gen (Hinh 4, gieng 3).

Nhiing the bien nap c6 kieu hinh Mut+ cho ket
qua kieu gen nhu du down.

op m6i 5'AOX1 —3'AOXI.
San pham PCR vbi khuon la be gen tir
cac chang Mut+ va cap mei 5'AOXI —3'AOX1
c6 kich thuac khoing 2200 by (china gen
AOX1) (Hinh 4, gieng 3) con tir chung Muts thi

Trang 49


Science ii Technology Development, Vol 15, No.T1- 2012
1"

Hinh 4. Dien di (Or sin phim PCR cha

Mid) 5. Dien di SDS-PAGE (1-4) va lai Western (5-8). 'Mang

pPICZaA/h-mpi (2), DNA bij gen P. pastoris

char' protein (1, 5); P. pastoris GS115::pPICZaA (2, 6); P.

(3), DNA by gen P. pastoris GS115::h-mpi có

pastoris GS115::h-mpi khong &Km (3, 7) va dirqc cam dug

kieu hinh Mutt (4), thang chili!' DNA 1 Kb (1).

methanol (4, 8).

Bleu hien MPI ngoai bko 6* P. pastoris
GS115::h-mpi

KET LUAN
Chung nem men P. pastoris GS115::h-mpi

Deng to bao P. pastoris GS115::h-mpi duce

da dircyc ceu truc thanh ding va bieu hien clusx

cam Crag bieu hien gen h-mpi beng methanol cO

protein muc tieu 6xHis-MPI clang ngoai bao.

vach protein yea kich thtrac khoang 8 kDa

Btrac deu duin nghiem kha !tang len men met de

(Hinh 5, giing 4). Cac mau doi chirng (Hinh 5,

cao bing he thong len men ttr &Ong BioTron-

gieng 2 va 3) khong they xuAt hien vach protein

LiFlus GX GX-05-12302 Cr quy mo 1 lit dat

nay. Sau CIO protein bieu hien (clang dung hop

ham ltrog khoang 178,4mg/I sau 96 gib len

6xHis-MPI) dtrot lai Western van khang the

men (Da lieu kh6ng trinh bay). Ket qua nay

dec hieu khang insulin HUI018 va phat hien

neu so van ket qua ducc Ong bo boi Jose M.

nhO khang the anti-mouse IgG-HRP. Vach lai

Pais ye Ong su (1,5g/1) van con the!). Tuy

Western (Hinh 5, gieng 8) co vj tri ttrong Crag

nhien, ket qua nghien ciru cue chOng toi co the

voi vj tri vach protein dtr doin la MPI-6xHis

cai thien sau khi tien hanh MIL sat cac dieu

(HIM 5, gieng 4). Mar vey (king P. pastoris

kien len men tei tru.

GS115::h-mpi da bleu hien MPI-6xHis ngoai

bao khi cam ang bang methanol.

Trang 50

TAP CHI PHAT TRIEN KH&CN, TAP 15, SO 11- 2012
CONSTRUCTION OF YEAST PICHIA PASTORIS EXPRESSING SECRETED MINIPROINSULIN INTO CULTURE
Ngo Thi Kim Hang, Vo Minh Tri, Tran Lind Thuoc
University Science, VNU-HCM

ABSTRACT: Diabetes is one of the diseases, which have recently gained significant attention. In
order to produce the recombinant insulin for treating diabetics, we have cloned and expressed a miniproinsulin in Pichia pastoris. The DNA fragment containing a gene encoding the fusion protein of
miniproinsulin-6xhis was cloned into the integrative plasmid pPICZaA resulting in pPICZaA/h-mpi to
construct the strain P. pastoris GSI 15::h-mpi expressing the recombinant MPI as secreted protein into
culture. Phenotype and genotype of P. pastoris GSI 15::h-mpi were confirmed by PCR and restriction
pattern. MPI was expressed as a fusion protein with 6xhis tag by supplementing methanol at a final
concentration of 0.5% in BMMY culture medium for 96 hours after inducing.
Key words: gene expression, mini-proinsulin, Pichia pastoris
TAI LIEU THAM KHAO
[1]. S. G. Chang, D. Y. Kim, K. D. Choi, J.
M. Shin, H. C. Shin. Human insulin

protein production using the Pichia
pastoris expression system, Yeast, 22,
249-270 (2005).

production from a novel mini-proinsulin

[6]. N.H. Cubing. Benh nOi tilt chuyjn hoa

which has high receptor-binding activity,

cicii thcio cluerng, Nxb Y Hoc Ha NOi

Biochem J. 329, 631-635 (1998).

(2002).

[2]. J. M Cregg, Vedvick, Raschke WC.

[7]. J. M. Pais-Chanfrau, Y. Garcia, L.

Recent advances in the expression of

Licor, V. Besada, Castellanos-Serra L,

Pichia pastoris.

Cabello CI, et al, Improving the

foreign genes in

Bio/Technology, 11, 905-910 (1993).

expression of mini-proinsulin in Pichia

[3]. R. Daly, M. T. Hearn, Expression of

pastoris, BiotechnoL Lett, 26, 1269-1272

heterologous proteins in Pichia pastoris:

(2004).

a useful experimental tool in protein

[8]. J. Sambrook, D. W. Russell, Molecular

engineering and production, J. MoL

Cloning, A laboratory Manual, Third

Recognit, 18, 119-138 (2005).

Edition, Cold Spring Habor, NewYork

[4]. International Diabetes Institute. What is

(2001).

diabetes?, Diabetes fact sheet (2004).
[5]. S. Macauley-patrick, M. L. Fazenda, B.
McNeil, L. M. Harvey, Heterologous

Trang 51