C C G N G NGHe SINH HQC T O A N Q U 6 C 2013
Bl6U HIEN V A T I N H SACH HORMONE SOMATOTROPIN B 6 Cf PICHIA PASTORIS X33
Ld Th| Hu$, Nguyen Th[ Thdo, Dong Van Quy^n, Quyen Dinh Thi Vidn Cilng ngh^ sinh hgc. Vi$n Hdn Idm Khoa hgc va C6ng nghd Vi^t Nam
T 6 M TAT
SomatotFopm I& mpt hormone sinh tmdng duyc ISng hpp boi cac t^ bao somatotroph 6 tuySn ygn cua bo va c6 boat tinh sinh hpc li
\ixn tang khi n^ng ti^t sira 6 bd cung nhu mpt sd v$t nudi khac nhu de. ciiu. Somatotropin bd taitd hop (rbST) Id san ptiim cdng ngh| sinh bpc dau ti£n da vd dang dugc sii di^ng rgng rai dl nSng cao ning suSt sua d t>d nhim dip ung nhu cSu ngiy cang ling cua n^on dinh dumg Trong bai b ^ n^y c h ^ g tdi da nghien ciiu biiu hien bST dang tu nhien (gdm 191 ammo acid) va d?ng bST cat bicn A9-bST (cat 8 ammo acid dSu N vii glycine 6 vj tri dfiu ti&i dugc thay b5ng serine) trong P. pastoris X33 vdi vector pPICZoA.
rbST vi A9-rbST deu dugc bi&i hi§n ngo^ IKIO va cd khoi lugng khoang 26 kDa, cao hon so vdi khdi lugng tinh Win ban d5u ih. 22 vi 20 kDa do bi glycosyl hda. Mure dg bi€u h i ^ ciJa rbST chi€m khoing 7,8% protein tdng sd ciia djch nuoi ciy ngoai bio, luong ducmg khoing 123 mg/L djch ouoi cay. Trong khi miic do bilu hifn ciia A9-rbST bieu h i ^ cao hon, chi6m khoang 35,3% protein ldng ngopi bio, tuong duong khoang 60 m§/L d|ch nuoi cfiy. ibST v i A9-rbST da dugc tinh s?ch khoang 98% qua c^t sic ky 4i luc Probond™Nickel-Chelaling Resin. H i ^ suat linh sjch rbST li 11,4% trong khi A9-rbST la 8,4%.
Tu khoa: Bilu hi?n, P. pastoris, hormone somatotropin bd, ibST.
Somatotropin b6 (bovine somatotropin hay bST, bGH- bovine growth hormone) Id hormone sinh tnrang dirge tim thiy ir gia sue. 06 4 d^rig hormone tu nhi§n, m6i lo^i la m6t chdSi polypeptide co chira 190 hoac 191 amino acid. Hormone dup'c tiSt ra d tuyen yen va i n h hu'dng din cdic hogt dQng cOa ccr the. Tnsng th£ri k^ c6 sira ir hit. somatotropin giiip huy dQng mcr ca thi, tang ndng luvng thu nh^n tCr thi>c Sn theo hijo^ng sdn xuit nhilu sOa hon so vdi ting tigp cdc md.
NhQng tic ddng cfia bST l§n gia sfic lln diu tien du^c Azimov va Krouze cdng b l ndm 1937, hai nha nghi§n cuu 3d cdng b l chit cd ho^t ti'nh tdch chilt tCr tuyin ydn cd tdc ddng Idm tdng sdn xuit sOa vd khdng dnh hudng din bd. Phdi din nhiing ndm 1980 nhd cdng nghd DNA tai t l hg'p mdi cho phep tgo bST tdi t l figp (rbST) d l cd t h i sdn xuit lijgmg Idn. HJ$n nay cd khodng 17% cdc con bd sCra ir My duvc ti^m somatotropin. Theo cac nghidn ciiu phan tich ddnh gid hi$u qud tidm bST trdn bd da cdng t i l , nhOng con bd dug'C tidm rttST lam tang san lu'grng sua tir 11-25% vd diat lugng thit bd, cdc thdnh phin trong sua, thjt khdng bj thay dli so vdi nhi>ng con tid khdng du^c tidm rbST. Tgi chdu A, rbST dd dug'C FDA chip nhdn Id sdn pham cdng ngh$ sinh hgc ndng nghldp diu tien trong cdc sdn phim cdng nghd sinh hpc ndng nghi§p vdo 11/1993 (Bauman, 1999) vd sua ti> nhung con bd duvc b l sung rbST Id sdn phim thyc pham si> dgng cfing nghd gene diu tidn dirg'c chlnh phu My cdng nh§n.
Somatotropin da duvc nghidn ci>u bilu hidn nhilu trong E. coli (Ctioi et al.. 1998, Choi etal., 1999, George e( a/., 1985) Bdc bi§t theo cdng b l cda Wingfield vd ding tdc gid (1987) da dua hai dogn DNA ma hda bST Id A-1 (dogn trlnh Xg day dii nhung amino acid alanine ir diu N dup'c thay bing methionine) vd dogn A-9 (doan bST dly dii bj d t 8 amino acid diu N vd glycine d vi trf diu ben dug'C ttiay bang serine). Kit qud A-9 bieu hidn rbST cao han vd tinh chat khdng thay dli so vdi A-1 vd bST ty nhidn (Wingfield et al.. 1987). Tuy nhidn, bST dug'C bieu hign trong E. coli thudng t i n tgi d dgng thi vCii, thudng gay trd nggi cho vide tinh sgch, hogt hda khdi phyc Igi dgng cd hogt tinh. Vi vdy, vdi vide khdng bj glycosyl hda, bST cd t h i du^c t)ilu hidn ngogi bdo trong P. pastoris d l thudn tidn trong qud trlnh tinh sgch dang c6 hogt tfnh. Theo nghidn cd'u cua Ouyang vd dong tac gdi, (2003), somatofropin ir ign (pST) dd duvc bilu hi|n virgt muc trong P. pastoris tuing vector pPICZoA vdi nang suit khodng 900 mg/L trong dilu kidn null Idc vd rpST khdng bj glycosyl hda (Ouyang e( al., 2003). Ndm 2003, Wang et al. da chdn cDNA honnone sinh hirdng ca chep (gcGH) vdo vector PG/VPZ- alpha-B dirdi sy dilu khiln cda promoter G/VP va bilu hidn thdnh cdng gcGH dgng cd hogt tinh, cd kich thudc 22 kDa gidng v^' homione ty nhidn vdi nang suit 50 mg/L (Wang e( al., 2003). Gatlando vd ding tdc gid (2003) da sOr dyng vector pPIC9 bilu bidn bST d P. pastoris GSl 15 thu duvc pratein 22 kDa, protein bilu hien ngogi bdo rit it. protein d l bi phan huy trong djch nudi cly, dudi « l u kidn quang hda. Ascado-Martinez va Ban^era-Saldaiia (2004) da bilu hien hormone sinh frudng chd (rOFGH) d chOng nam men Mut^ vd chung Mut*, protein ting so dgt tuong i>ng 40 pg/ml vd 15 Mg/ml djch nudi. Ham luvng rCFGH bilu hidn d chung Mut® Id 24 pg/ml, chilm 60% protein ting sd, cao hon g i p 8 lan so vdi bilu hidn d chfing Mut* vd c6 hogt tlnh tdng sinh khi thi> nghidm dli vdi t l bdo Nb2 (Ascacio-Martinez, Ban-eia- Saldaria, 2004). Santos et al. (2009) trong patent cdng b l da t l i i/u codon gene bGH vd si> dung vector pPICZoA bilu hidn d P. pastoris X33. BGH bilu hidn cd kfch ttnsifc 22 kDa, ddng bilu hien bGH cao nhlt cd ham lug'ng bGH 29,39 mg/L, mdc dg bilu hidn chilm 72,4% protein dich ngogi bdo (Santos e( a/., 2009).
Trong bdi tido ndy, chung tdi nghidn ciiu bieu hidn bST, A9bST d nim men Pichia pastoris X33 d l tiln tdi san xuit vd thi> nghidm ndng cao nang suit slJa d bd si>a.
VAT Lieu VA PHUtyNG P H A P Vgt li#u
Bogn gene ma hda protein somatoti^pin dly du bST da nhan ddng trong pCR2.1 duoc dfing Idm khudn d l khuich dgi dogn gene dSTvd A96STvdi cdp mil dgc hidu phu hg'p dua vdo pPlCZoA.
Plasmid pJET1.2 (Fermentas) dug'C dung d l nhdn ddng vd pPICZoA (Invitrogen, M^) diing d l bilu hidn gene dSTvd
H(?l NGH! KHOA H p c CONG NGHE SINH H(?C ^ " " " i P l ^ - ^ . , / H
A9bSr.
Chiing E. coff DHIOB (Invitrogen. My) dugc si> dyng d l chpn ddng cac plasmkl ldi t l hgp. Chung P. pastoris^^
(Invitrogen, My) du?c su dyng d l bilu hidn gene bSTvd ASbST.
Khdng t h i khdng bST (anti-bST) san xuit trdn thd do phdng Vi sinh vgt hpc phdn ti>, Vidn Cdng nghd sinh hpc cung dp.
Mil khuich dgi gene bST dupc thilt ke dya vdo trinh tv cDNA cOa bST da cdng b l trwi Genbank: pP_WT_bST_F: S- gcgaattc gccttcccagccatgt-3- vd pP_bST_R: 5'- gctctagagcgaaggcacMdggo^-; pP_D9_bST_F: S- gcgaattcagcctgtttgccaacg-3' vd pP_bST_R: 5'- gctctagagcgaaggcacagctggc-3'. Mdi 3'AOX1 (5"- gga tgt cag aat gcc an tgo-3') vd 5'AOXI (5'-gac tgg ttc caa ttg aca agc-3') dOng d l kilm tra sy hdi nhdp cda gene bST vao genome cda P.
Mdi tnrdng nudi cay LB kw salt gim 1% peptone; 0,5% yeast extract 0,5% NaCl; pH7-7,5. Mdi tardng nudi d y LB low salt ddc dupc b l sung 1,5% agar. Mdi trudng YP (1% cao nim men ; 2% peptone) cd bd sung glycerol 1% d l nuA preculture t l bdo nim men.
Phuong phap
PCR khu4ch a^i gene bST vd ASdST: Gene bST. A9bSr dupc khuech dai tfr khudn pCbST vdi cdc c$p mli d§c h($u tuong dng: pP_WT_bST_F vd frf'_bST_R; pP_D9_bST_F vd pP_bST_R d l ttiilt k l chen vdo pPICZoA. Hon hgp ph4n img PCR 25 pi gbm 0.5 pi DNA (-100 ng); 1 pi mli xudi (10 pmol), 1 pi mli ngupc (10 pmol); 2.5 pi MgOtz (25 mM); 2,5 Ml ddm lOx PCR; 2 pi dNTP (2,5 mM); 0,25 pi Taq DNA polymerase (5 U/pl); 14,25 pi H2O. C h l dd chgy: 95°C/4 phiit;
35 chu ky (95<^/30 gidy; 55=045 gidy; 7200/45 gidy); 72''C/10 phiit.
Cit enzyme gidi hgn vd finh sgch: vector bilu hi#n pPICZoA vd gene bST (hodc gene A9bST} dupc cit bing EcoRI vi Xbai trong phdn i>ng gim: 5 pl ddm lOx tango; 1 pi (10 U) EcoRI; 1 pi Xbai (10 U); 15 pi sdn phim PCR dogn dSTho|c A9bSr, hodc pPlCZoA; 23 pi H2O. 0 phdn dng d 37'0 trong 3 gid. Djch cit sau dd dupc chgy didn di frdn gel 0.8%
agarose vd dupc tinh sgch tilrtg kit Agarose Gel Purification (Qiagen) theo chl d§n cua hdng.
Phdn Ong n6i ghdp: Hai dogn gene bST, &9bST vd vector pPICZoA md vdng sau khi tinh sgch dupc nli ghdp vdi nhau nhd TA ligase d 22'C (tfr 2-4 gid) trong hin hpp phdn yng nli ghdp (gim 1 pi ddm lOx phdn dng; 0.5 pt (5 U) T4 ligase;
2 pi dogn chdn 6ST (hodc A96S7); 2 pi pPICZoA; 4,5 pi H20) d l tgo thdnh plasmid tdi to hpp tuong dng Id pPA9bSTvfl pPbST. Plasmid tdi to hop dupc biln ngp vdo t l bdo khd biln £ cdi DHIOB ^ e o phuong phdp sic nhi$t vd sdng Ipc frdn mdi trudng LB k)w salt cd b l sung zeocin 25 pg/ml.
Bi4n n^p plasmid vdo P. pastoris X33: plasmid pPAObST vd pPbST dupc d t md vdng vdi Saci trong phdn ilng gim: 10 pi d$m lOx tango; 3 pi (10 U) Sad; 30 pi pPbST hogc pPAStiST; 57 pi H2O; li phan dng a 37°C trong 3 gld. Plasmkl md vdng sau dd dupc tinh sgch bing kit tinh sgch sdn phim sau phdn ung d t (Reaction deanup, Qiagen) theo chl din ci:ia hdng. Tilp theo, plasmid tdi t l hpp da md vdng dupc thjn diu vdi t l bdo khd bien vd chuyin vdo cuvette xung (K$n, <A frong dd khodng 15 phuL Mlu dupc xung didn vdi dilu kidn 2 kV. 25 pF, 400 Q, sau dd b l sung ngay 1 mM M sorbitol dd lam Ignh trudc vd d l trong da khoang 15 phiit. Chuyin todn bd djch biln ngp sang Ing ^Icon 15 ml vd Cl d 30°C trong 1-2 gld. Djch biln ngp sau dd dupc trdi frdn dta mdi tardng YPGS ddc cd b l sung 100 pg/ml zeocin, it ir 30'C trong 3-5 ngdy Khuin Igc mpc ti#n dia chda gene khdng zeocin dupc nudi trong mdi trudng YPG b l sung 100 (jg/ml zeocin d l tdch ONA kilm tra gene chdn.
Cdc ddng P. pastoris X33 tdi t l hpp c6 gene bST. ASdST hdi nhdp vdo dimg vj frf AQX1 trong genome dupc chpn lpc bing phdn dng PCR vdi khudn DNA ting s l tdch chilt tu cdc ddng vdi cdp mli 3'AOX1 vd 5'A0X1 trong phdn 25 ii gim: 0,5 pi DNA (-100 ng); 1 pi mil xudi (10 pmol), 1 pi mli ngupc (10 pmol); 2,5 pi MgCl2(25 mM); 2,5 pi ddm IO1 PCR; 2 pi dNTP (2,5 mM); 0,25 pi Taq DNA polymerase (5 U/pl); 14,25 pi H2O. Chl dg chgy: 95''C/4 phut; 35 chu k*
(9500/30 gidy; 54°C/45 gidy; 72°0/45 gidy); 7200/10 phut. San pham PCR duoc didn di trdn gel 0,8% agarose d l Idlm tra.
Biiu hidn rbST vd r&9bST tn>ng P. pastoris X33: Nudi d y mpt khuin Igc PXbST va PXA9bST (chCing P. pastoris X33 d genome da hdi nhdp plasmkl pPbST vd pPA9bST) frong 5 ml mdi frudng YP cd b l sung glycerol 1 %, nuBi qua dfim 6 30"C. lie 200 rpm. Khi mgt dd t l bdo OD600 dgt tfr 2 din 6. chuyin gilng sang mdi trudng YP cd b l sung methanol 1*
d l dgt dirpc ODeoo = 1. Sau dd. tilp fi,ic nu« lac 200 rpm d 28 - 30''C. T l bdo dupc cam dng bing methanol sau mli 24 gid vdi ning dd cull cdng 1% Djch nudi d y sau 3 ngdy cdm Crng dupc do pH, OD600 vd dem ly tdm 6000 ipm trong 10 phiit d l thu djch trin chCra protein ngogi bdo vd tiln hdnh xdc djnh protein bing phuong phdp didn di trdn gii polyacrylamide 12.5% vd do Bradford xdc djnh hdm lupng protein ngogi bao.
77nh SQch^pmtein tdi t6 hgp: rbST va rA9bST bllu hidn frong djch ngoai bdo dupc tinh sach bing cdt sic ki & \\/c Probond Nickel-Chelating Resin. Protein tinh sgch dupc kilm tra frdn gel 12,5% polyacrylamide. Hdm lupng protein dupc xdc i^nh bdng phuong phdp Bradford (1976).
Nhdn di$n bST tdi td hgp: Tinh ddc hidu cua bST tai t l hop duvc xdc djnh bang phdn dng lai Westem giua bST v«
khdng thd khdng bST san xuit d frid. bST tdi t l hpp cung dupc nhdn dien bang MALDI-TOF.
K f r QUA V A B I ^ N LUAN
Thiet ke vector bilu hifn bST va A9bST trong P. pastoris j Doan DNA ma hda protein bST dly du ddi 573 bp vd A9bST ( d t 8 amino acid diu Nl ddi 549 bp dupc khuich d?!Ill
D J'e-P o ^ J l i L.^^"" P'^^'ST bdng cdc cgp moi tuung ung pP_WT_bST_F va pP bST_R; pP_D9_bST_M pK_bbr_K. Ket qud Hinh 1 cho thay dogn gene bST (Idn 2) vd A9dST{lan 1) da duoc khuich dgi dac hidu. Sdn ph*"
- : : C 6 N G NGHE SINH H p c T 0 A N Q U 6 C 2013
ndy dupc dung d l dua vao pPICZoA.
Gene bST, &9bST duvc d t vdi EcoRI vd Xbal vd dua vdo plasmid pPICZoA cung dupc md vdng bang EcoRI vd Xbai tgo fridnh plasmid tdi t l hpp pPbST vd pPA9bST. San phim d t kilm tra plasmid tdi t l hop pPbST vd pPA9bST vdi EcoRI vd Xbal ir Hlnh 2 cho thiy cd hai t>dng, mdt bang cd kich ttiudc khodng 3600 bp (hrong ung vdi kich ttiudc plasmid pPIOZoA) vd mpt bdng cd kich thudc khodng 600 bp (tuong i>ng vdi gene bST vd A96ST). Kit qud PCR vdi khudn plasmid tdi t l hpp pPbST va pPA9bST (Hinh 2. Idn 5 vd 6) cOng cho ttiay sdn phim PCR vdi cap moi AOXI frong placid pPICZoA c6 kich ttiudc -1200 bp. dung vdi kich ttiudc Unh todn cua dogn gene bST vd A9bST (-600 bp) cung vdi dogn signal peptid frong pPICZoA (-600 bp). Nhu vdy, gene dSTva A9bST da duvc chen thanh cdng vdo pPICZoA vd ttiu dupc plasmid tdi t l hpp pPbST, pPA9bST tuong yng d l bilu hi#n frong P. pastoris.
Hinh 1. Ellen dl d l sdn phim PCR khuech dai Hlnh 2. Dl^n di sdn phim d t plasmid m t l hgiJ pPbST (3-4), pPiSbST gena bST (2) vd A9bST (1) v6l c$p mil (1-2) bing EcoRI vd Xbal vd sdn ph£m PCR tir khudn pPbST (6) vd pP_WT_bST_F vd pP_bST_R; pP_D9_bST_F pPA9bST (5) vM cip mil AOXI, dll chi>ng HjO (dc), thang DNA 1 kb v i pP_bST_R; thang DNA 1 kb chuin (M). chuin (M)
Mdt khdc, plasmid tdi t l hp|j pPbST, pPAObST sau dd cdng dupc dpc frinh ti/ vdi mli 3'AOXI d l kilm tra Igi khung dpc Kit qud dpc trinh ty cho thay gene bST vd A9dST dd chdn vdo diing vj fri EcoRI - Xbal ctia plasmid pPICZoA vd ddng khung dgc md.
Biln ngp plasmid pPbST vd pPA9bST vao P. pastoris X33
Plasmid tdi t l hpp pPbST vd pPA9bST dupc d t md vdng vdi Sad vd dupc ttnh sgch Igi qua kit. Kit qud Hinh 3 cho thiy pPbST vd pPA9bST da dupc d t md vdng hodn todn vdi mOt bang duy nhlt cd kich ttiudc khodng 4200 bp, ddng vdi kich ttiudc cCia gene dSTvd ASbST (-600 bp) d n g vdi plasmid pPIOZoA (-3600 bp). Khudn plasmid tdi t i hpp md ving ndy dupc ddng d l biln ngp vao P. pastoris X33.
Plasmid tdi t l hpp pPbST vd pPA9bST md vdng dupc biln ngp vdo P.
pastoris X33 theo phuvng phdp xung didn, tgo thdnh cdc ddng tdi t i hpp tuong li-ng Id PXbST vd PXA9bST. Cdc ddng P. pastoris X33 tdi to hpp dupc chgn Igc trin mdi trudng YPGS cd b l sung 100 pg/ml zeocin. Sau 3-5 ngdy 0 d 30°C, cdc ddng cd sy hdi nhgp cua plasmid tdi t l hpp vdo genome se cd khd ndng khdng zeocin vd mgc frdn mdi trudng cd bo sung zeocin.
Cdc ddng mgc trdn mdi trudng chpn Igc dupc nhdt nudi d l tdch DNA ting s l vd kilm tra PCR vdi cgp mil AOXI. Kit qua cho thiy sdn phim PCR vdi cdp mli AOXI frxing plasmid pPICZoA cd Rich ttiudc -1200 bp, diing vdi kich thudc tlnh todn cua dogn gene bSTvd &9bST (~600 bp) Cling vdi dogn signal peptid trong pPICZoA (-600 bp) (hlnh
4). Ngodi ra sdn phim PCR cdn cd bdng -2200 bp, ddy Id bang sdn pham phdi cd trang h$ gene cCia chOng P. pastoris.
Nhihig ddng cd san phim PCR xuit hi§n ding thdi ca hai bdng (m$t bdng d u tnic plasmid dua vao -1200 bp vd mdt bdng -2200 bp) Id nhung ddng cd sy hii nhdp ddng ciia d u biic gene ngogi lai. Nhu vdy, cdc ddng 3, 4. 5. 6 bilu hidn bST (PXbST) vd ddng 8-18 bilu hidn A9bST (PXA9bST) (Hlnh 4) Id nhung ddng tdi t l hpp ttidnh cdng gene bST vd A9bSTvdo P. pastoris X33.
Hinh 3. Dl#n dl d6 sdn phim plasmid pPbST (Idn 1-2) va pPA9bST (ldn 3-4) sau khi cat tn&
v6n^ vdl Sad va tlnh sgch; thang DNA 1 kb chuan (M).
Hlnh 4. Difn dl sdn pham PCR ktim tra sy h^i nh^p ciia clu tnic bleu hifn pPbST (1-7) vd pPA9bST (8-17) vdl c$p mil AOXI Bllu hifn bST vd A9bST d- P. pastoris X33
Cdc ddng PXbST vd PXA9bST dupc nudi bilu hiln mdi trudng YP cdm irng methanol 1% sau mli 24 gid. Sau 72 gid cdm dng. dich nudi dupc ly tdm thu d|ch ngogi bdo d l phan tich bang SDS-PAGE nhu md td tren Hinh 5. Theo tinh todn IJ ttiuylt protein bST vd A9bST cd kich thudc khodng 22 vd 20 kDa. Tuy nhidn, kit qua phdn tich SDS-PAGE frdn hinh I • cho thdy d cdc mlu PXAObST (Idn 2-5) vd PXbST (Idn 6-8) xuit hien bdng kich thudc khodng 26 kDa md d Idn 1
HOI NGH! KHOA HQC C O N G N G H E SINH HOC 7 ^ - i'4_ t ! i l L ' l U J . l ! ? ^ l ^ ^
(miu dli diimg dm pRczaA/X33) khdng cd. Bay cd ttil td bdng bST vd A9bST cd kich ttiudc Idn hon so vdi tlnh ^ n H do kit qua cua qud frinh glycosyl hda d t l bdo nhdn chuan. Ket qua nay khac vdi dy dodn ban ddu khi sCr d y i a p ^ mim phdn tl<3i glycosyl hda vj fri O (Net OGIyc 3.1 Server) vd N (NetNGIyc 1.0 Server) hdn frinh t y amino aad ( A ^ d i u cho k^ qua dy dOan khdng bi glycosyl hda. Ngoai ra, kit qud nay cOng khdc vdi mot so cong b l bieu hidn hornnl sinh frirdng bd vd mgt so vgt nudi khdc nhu Ipn, cd d P. pastoris diu khdng bi glycosyl hda. Jing Ouyang e( a/, (20^
bilu hien hormon anh frudng ciia Ion pST, Wang ef al. (2003) bilu hign honnone sinh frirdng cd chep (gcGH), G a M e( al (2003)- su dung vector pPIC9 bilu bidn bGH d P. pastoris GSl 15; Neiva Sanhis ef al. (2009) sau khi t l i uu ^&
gene bGH vd su dung veCtor pPICZoA bilu hien d P. pastoris X33 deu cho ket qud Fmtein khdng bj glycosyl hda. n W Trong cdc ddng thu dupc, dmg PXA9bST1 (Idn 2) va PXbST3
(Idn 8) cd bang 26 kDa dgm hon cdc ddng cdn Igi. Kit qud phan tich ddnh gid muc dd bieu hidn protein tdi t l hyp bang phin mim Dolphin ID cua hai ddng ndy cho thay rA9bST chiem 35,3% protein ngoai bdo (tuong duong 60 mg/L djch nudi d y ) , cao hon 5 lan so vdi rbST chilm 7.8% (tuong
Hlnh 5. Difn di SDS-PAGE djch protein ngogi bte Uiu du'Q'c t£r dich nuoi cly chiing n£in men t^ t6 h^p pPiczaA/X33 (lan 1), PXA9bST (Idn 2-5), PXbST Qiu 6-S);
thang prolein chuan (M) duong 12,3 mg/L djch nudi d y ) . Nang suit bilu hien rbST
thap hon so vdi mdt s l cdng bo bilu hign somatotropin trong khi rA9bST c6 nang suit IMIU hign tuong duong hogc cao hon so vdi cdc cdng tid cua Wang et al, (2003). bilu hidn hormone sinh frudng cd chdp (gcGH) vdi ndng suit 50 mg/L; Ascasio- Martine et al. (2004), bilu hign hormone sinh tnjong did (rCFGH) a chiing Mu^ chian 60% protein ting s6, fcjvng duong 24 pg/ml. Nehra Santos ef al. (2009), bilu hidn gene bGH sau khi t l i uu hda codon cd ndng suit 29,39 mg/L (chilm 72,4% pratein ting s l cua djch ngogi bdo). Tuy vdy ham lupng somatotropin tdi t l hgp thu dupc nang suat cdn rat thap
so vdi cdng b l ciia Ouyang ef al, (2003) bilu hign somatotropin Ipn (pST) d P. pastoris X33 vdi nang suit 900 mg/L. Do dd, cdc dioi kifn nudi d y se cdn dupc tilp tgc t l i uu d l tang hidu qua bilu hign rbST.
Tinh sgch, nhan dien bST va ASbST tai t l hpp
Hai ddng PXA9bST1 va PXbST3 (Hinh 5, Idn 2 vd 8) dupc chgn nudi bilu hign somatotropin d binh tam gidc 250 ml trang 50 ml mdi frudng YP d m ung mettianol 1% ci> sau moi 24 gid, sau 72^ gid nudi d m dng mdu dupc ly tdm ttiu d\ch nli d l kilm fra sy cd mgt cua protein tdi t l hgp somatofropin. Protein tai t l hpp bST vd A9bST bilu hign ngogi b^
dudi dgng gan thdm 6-histidine ir d i u N, vi vay protein tdi t l hpp de ddng duyc tinh c h l bang cdt sac ky di li/c v*
histidine. Sau khi tinh chl, djch tnh sach d cac phan dogn dupc dign di tren SDS-PAGE va nhudm bgc (Hinh 6).
M 1 2 3 4
Hlnh 6. SDS-PAGE protein roMiST: djch len cot (1) cac phan doan 1 3 sau Hinh 7. Westem blot phan tich phdn dng gii^
tinh5gch{2-4);va rbST: djch len cot (10), cdc phan dogn 1-5 sau tmh sach (S- khang the khang bST vdi phan doan riStS 9); thang protein chuin (M) tinh sach (1), thang protein mdu chuan pul) Kit qua frdn Hinh 6 xuit hien mdt bdng kich
ttiudc khoang 26 kDa d phan dogn 1-3 (idn 2-4) ciia mdu PXAbST vd phdn dogn 1-2 ciia mau PXbST (lan so 5-6). rfaST vd rA9bST thu dupc cd kich thudc khoang 26 kDa, dung vdi bang dy dodn Id rbST vd rA9bST bilu hidn bj glycosyl hda ngn cd khli luong cao hon ban dau. rtiST vd rA9bST dupc tinh sgch khodng 98% (theo ddnh gid cua phan mim Dolphin 1D) vd higu suit linh sgch rA9bST dat 8,4% va rbST Id 11.4%.
Mau dich pratein ngogi bao thu dupc tCr dich
nuoi cly chung nim men tdi to hpp Hlnh 8. Khoi phi d6ngvj ciia chuoi peptide NYGLLSCFKK tai vj trf 157-166 PXA9bST1 dupc dem phan tich kha nang *="^ SOMA_CRONO tren Swiss-Prot theo diem ban ph^ ion 61 va p<0,05 lidn kit vdi khdng thi ddc higu khang bST.
Kit qud phan tich phdn Cmg gii>a khdng the khang bST vdi d|ch protein rA9bST tmh sach chl thiy xuit hign mgt bSnfl:
duy nhat kfch thudc khodng 26 kDa dung vdi kich ttiudc rA9bST (inh sgch (hinh 7). Nhu vgy, bdng protein kich thif*
KK ~ gj l^f. 1 ^ , , 1
• iV
i \ 1 G/
1!
J50 ' W 7^ iOOO 12»
: C O N G N G H E S I N H H Q C T O A N Q U O C 2 0 1 3
2 6 k D a d i i n g Id s o m a t o f r o p i n t d i l l h p p vd khi b i l u hien d r»lm m e n d a bj glycosyl h d a n d n kich ttiudc tdng ldn 2 6 k D a . W t k r ^ c b d n g p i y i e i n rASbST l i n h s g c h d H i n h 6 (lan 2 ) c u n g d u p c d t d l p h d n t i c h k h l i p h i M A L D I - T O F . K i t q u a c h o ff^dogn p e p t i d e N Y G L L S C F K K c u a rA9bST tinh s a d i g i i f g v d i S O M A _ C R O N O ( 1 5 7 - 1 6 6 ) ( H i n h 8 ) , S O M A _ M U S V I ,
!>UMA_13ALB0 frdn S w i s s - P r o t N h u vdy, b S T d a d u p c bieu hidn ttidnh cdng d P. pastoris.
KtVLUAM
C h u n g toi d a b i l u hien v d t n h s g c h ttidng c d n g r t i S T v d rA9bST d n a m m e n P. pastoris X 3 3 . N d n g s u i t b i l u hien ciia W N k " ^ ^ '•'iSt^ST Id 6 0 mg/L d j c h nudi t n l u h i d n . r b S T v d rA9bST d u v c tinh s g c h 9 8 % q u a c d t d c k y frao
UAciman
f^^'V^'diduyKlhve hjfnvAi nguAn lankpU kS trp tir di ldi '•N^ain cOu qigi trinh oong nghf son xudi hormone somatotropin few Id hgp nhdm tdng auiaiStsmabasua . Bp Ndng nghi^ vd phdi tnen Nong Am (Chimg Irirdi ir^g ^im mg dvng cdng nghf sinh hoc irong linh vyc nong nghi^
vaphai tnen mmg Ihon den nam 2020).
T A I L i e u T H A M K H A o
Choi, JW, Kim SI, Lee SY (1998). Purification and c^araderization of recombinant bovine growth homione produced in Escherichia coli.
BiotBchnology Letters 20: 269-273.
Choi, JW, Ra KS, Lee YS (1999). Enhancement of bovine growth hormone gene expression by increasing the plasmid copv number.
Biotechnology Letters 2V.^-5. J a •- ™ K J George, HJ, f i t a l i e n JJ, PItadnski WP, Glassman DL, Kizyzek RA (1985). High-level expression In Escherichia coh of biologically active bovine growtti honnone. DWA 4:273-81.
WingliekJ, PT, Graber P. BueH G, Rose K. Simona MG. Burleigh BD (1987). Preparation and characterization of bovine gnwrth honnones produced in recombinant Escherichia coli. Biocdiem J 243:829-39.
Ouyang, J . Wang J , Deng R, Long Q, Wang X (2003). High-level expression, puriftaation, and diaradenzation of porcine somatofropin m Pichia pastoris. Pmtein EjqvPurifZZ: 28-34.
Ascacio-Martinez, JA, Banera-Saldaiia HA (2004). PnxlucQon and secretion of bwlogically active recombinant canine growth homione by Pichia pastoris. Gene, 340:261-266.
Santos, N, Ramos S, Teixeira D, Moraes P, Neto C, Almeida T, Torres A, Felipe S (2009).Production of recombinant Bos taunis growth homione with Pichia pastons. In IVorfd Intellectual Property Organization. Austna.
EXPRESSION AND PURIFICATION OF BOVINE SOMATOTROPIN IN PICHIA PASTORIS X33
Le T h i H u e , N g u y e n T h i TTiao, D o n g V a n Q u y e n , Q u y e n D i n h T h i Institute of Biotec^tonogy. VAST
SUMMARY
Somatotropin is a growtti honnone produced by die somatotroph cells in the bovine pituitary gland, and its biological activity is likely to increase milk production in cows as well as other animals such as goat and sheep. Recombinant bovine somatotropin (rbST) is Ihe first biotechnology product has lieen widely used to improve milk production in cows to meet the growing needs of nutntion
!D this p3|)er we studied expression of the natural bST fcnm (including 191 amino adds) and the modified bST fonn - AA9bST(the fijU-length twvine sequence is truncated at the N-terminus by eight residues and and glycine in Ihe liist position is replaced by serine) in Pichia pastoris X33 with pPICZoA vector. The rbST and AA9-rbST were expressed extracellular and their molecular masses are 26 kDa, higher than originally molecular mass of 22 and 20 kDa due to glycosylation. Expression level of itST was about 7.8% of the total extracellular proteins, equivalent to 12.3 mg/L culture. Whereas ifae AA9-rl>ST had a higher expression level, about 35.3% of toiai extracellular proteins, equivalent to about 60 mg/L of culture. The Ad9-rtiST and rbST were purified approximately 98% by affinity chromatography using Probond"*Nickel-Che!ating Resin. Purification efficiency of tbST was 11.4% while AA9-it)ST was 8.4%
Keywords: e;q)ression, P. pastoris, bovine somatotropin honnone, tbST
