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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 21 thcing 03 nam 2011 holm chink stia chat, 09 thcing 04 nom 2012) TOM TAT: Dai thao dwang la mat nhiing can benh clang nit chivy span tam hien 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 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 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 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 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, Benh DTD, chitin khoing 60-70% 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 cac mac Oat trien va 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 insulin c6 hoat tinh 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 citing n6ng d6 [1] [6] VI the, viec san xuat insulin ngutri bang Pichia pastoris la he thong bleu hien protein &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 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 qua trinh len thay vi 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 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 'PHTI Xho va '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 plasmid tai to Plasmid pMZZIn CO ngu6n g6'c to plasmid pUC 19 mang doan gen h-mpi dtrqc dung lam khuon 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 'AOX1 chu ky (94 C/1', 55 C/45", 72 C/2'); 72 C/ l0' Cap moi 'PHTI Xba (GTA CTC TAG ATT AGT TAC AGT AGT TCT CCA G) va '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 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 vai trinh tir 19 thuyet bang pilaf.' mem Jellyfish mM va -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 ding 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 tien sy Peer Nobert Jorgensen (Novo Nordisk, dung thing Sau dO, plasmid 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 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 cac the bi&I nap lam Ichuon PCR thu nhan gen h-mpi bang dugc kiem tra kieu hinh dua tren su ph& triAn cap m6i 'PHTI Xho va '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 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 30 C qua dem cho 5'PHTI-Xho va 3'PHTI-Xba (bit Op dec hi0 den OD580 dat 2-6 Ly tam 4000 rpm, ten gen h-mpi) phut a C Chuyen sang 200 ml BMMY de cam Ong su bieu hien protein num tieu (0D580 = a thai diem h) Sau moi 24 gib b6 sung Trang 47 Science & Technology Development, Vol 15, No.T1- 2012 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 Dien di san phim PCR gen h-mpi tir plasmid pMZZIn (2), thang chair DNA kb (1) Hinh Dien di san phim PCR cila pPICZaA (2), pPICZaA/h-napi bing cap moi 'AOX1_3'AOX1 (3), pPICZaA/h-mpi bAng cAp moi '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 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, 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 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 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 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 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 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 MMH IA mai throng china gen h-mpi xuat hien vach DNA c6 kich thtrac methanol lam ngutin carbon 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' 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) tir chung Muts thi Trang 49 Science ii Technology Development, Vol 15, No.T1- 2012 1" Hinh Dien di (Or sin phim PCR cha Mid) 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 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 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 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 va 3) khong they xuAt hien vach protein LiFlus GX GX-05-12302 Cr quy mo lit dat 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 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 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 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 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 ... 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... 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... 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
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