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Tap chi Cong nghe Sinh hpc 13(3): 821-830, 2015 KHAO SAT QUY TRINH TINH CHE NHAN TO TANG TRYONG Ti! TIED CAU rhPDGF-BB TAI TO HIP An V6 Kha Thanh Thanh, Nge Thi Huyen Trang, Nguyen Trf Nhan, TrAn Linh Thtrerc, Deng Thi Phtrang That) Truing Dgi hpc Khoa hoc nhien, Dai hoc Quac gia Thanh Ho Chi Minh Ngity nhan bai: 07.12.2014 NO), nhAn dang: 15.9.2015 TOM TAT Nhan to tang twang tin tieu eau ngtrai dong BB tai t6 hqp,(rhPDGF-BB, recombinant human Platelet Derived Growth Factor BB) di duqc dung tri lieu nhieu loci benh, d6 ph6 hien vi quan nhat la dieu tri 16 boa chin bien chimg cis benh dii thao duang Truac day, clic he thOng bleu hien nhu Escherichia coli va Saccharomyces cerevisiae di duqc img dung san xuat rhPDGF-BB Tuy nhien, vii nhieu tru diem wort fret, nam men Pichia pastoris dang duqc xem 111 met he tilting day tiem nang Ke than c8ng trinh can Vuong Cat Khinh va dang tic giA (2014) ye viec too c6ng chitng P pastoris tai to hip da ban bleu hien mph protein hPDGF-BB, nghien dm nay, chang tai tier, hAnh khao sat quy trinh thu nhin vi tinh the rhPDGF-BB a quy and phong thi nghiem Djch protein tong china rhPDGF-BB sau nu6i cay se duqc khio sat thu nhin bang phuang phip ttia amonium sulfate bao h6a vi phuang phap lqc tiep tuyen vii cet lqc kich thuac 10 kDa Protein mqc tieu dupe Bac, sat tinh cite bang phucmg phap sac ki trao dai cation va sac ki kj mac Kit qua cho they hieu suit thu nhan bang phucmg phi!) tua vii 60% amonium sulfate bio hoa vi iQc tiep tuyen a t6c dOc 125 vong/phat dat tuang img khoang 82% va 63% Quy trinh tinh cite bang phuang 010 sac ki trao doi cation dot hieu suit cao han phucmg phi!) sic ki kj ntrac, va duqc toi tru hoa dieu kien dung djch can bang va dung djch ii giii fat qui dung djch can ban& duqc chip IA dem acetate 20 mM pH4 va dung dich li giai duqc chin IA NaCl I M/Tris-HCI 20 mM pH7,5 de dot hieu suit tinh the cao nhat IA khoing 22% vii de tinh soch 99% a Ttlr kh6a: Lpc tiep tuydn, rhPDGF-BB, sac ki kj nmk, sac ki trao doi cation, nia amonium sulfate MO DAU Nhan to tang truing tin tieu cAu (PDGF) la met nhan to phan bao chit yeu cua nguyen bao scri, b#o ca tran va nhieu to bio khic, duqc san xuat tin tieu cAu (Heldin, •Westermark, 1999) Protein tic deng den nhieu to bao lien quan den qua trinh han gan vet thucmg nhu: kich thich su bao va huong hea to bao ca tran nguyen bio sqi va te (Ricardo et al., 2006), kich thich dai thuc bao san xuat va tiet nheng nhan to tang truing quan (Glenn et al 1991), kich thich su san sinh nhimg phin tir fibronectin (Blatti et al 1988), collagen (Lepistoe et al 1992) va proteoglican (Schenherr et al., 1991) Dua tren chine nang nay, protein PDGF ngtrai tai to hp clang BB (rhPDGF-BB) da clop Cmg dung de dieu trj 16 loet china benh nhan bj bien chimg au duong (Regranex® gel, HealthPoint, Fort Worth, TX, Mk) (Embil, Nagai, 2002) Protein rhPDGF-BB di duqc nghien tiro san xuat beri cac he thong bieu hien nhu vi khuan Escherichia coli (Karumuri et al., 2007), nAm men Saccharomyces cerevisiae (Wang et al., 2009) Trong nghien tiro bieu hien tren h¢thing E coli, monomer rhPDGF-B djch tan the viii dirge tinh such bang 110 thong sac ki long cao ap dao pha (RP-HPLC) sir dung cet Source-30, sau d duqc tai gip cuen va lqc qua amicon 10,000 MWCO, protein rhPDGF-BB thu duqc vii de tinh sach han 95% Doi vii nghien ciru tren hg timing S cerevisiae, djch nuoi cay china protein muc tieu duac tinh sach so be sau d6 duqc tinh the qua hai phuang phap sac ki la trao doi cation (sir dung cet CM-Sepharose) va lqc gel (sir dung cet Sephadex G-75), hieu suit thu heti cua ca qua trinh tinh che dat 20% Ngoai ra, gan day met so he &King mai ciing d A duqc ap citing nhu nam An Pleurotus eryngii (Choi et al 2011) hay cay thuec la Nicotiana tabacum L (Deepa et al., 2013) Protein tai to hip thu duqc djch chiet nam An c6 china duel His-tag, de dirge tinh che qua cet sac ki luc (cot Ni-NTA Spin china Nikel), nhien phuang phap nay, protein khong in clang to nhien ma 821 VO Kha Thanh Thanh et al chira His-tag nen can them cac nghien cau kiem tra tinh an Man hPDGF-BB cung di dirge bieu hien cong bang each chuyen gen tren thuc vet vi sin phim tinh sach cung di duqc chung minh c6 tic dung china lanh vet thuang in vitro Val nhi'eu uu diem Yucrt trei, nem men Pichia pastoris hien dang duqc xem la met he Meng day tem ning (Darby et al., 2012) Tuy nhien, viec sin xuit rhPDGF-BB dua tren he thong P pastoris van chua dirge cling bo Tai Viet Nam, Vuang Cat Khanh va deng tic gia (2014) di too thinh cong clang nem men P pastoris X33 tai to hqp mang 16 ban gen ma h6a protein hPDGFBB Trong nghien eau nay, ching Col tiep tic tien hanh khao sit quy trinh thu nhan va tinh che rhPDGF-BB a quy m6 ph6ng thi nghiem sir dyng chimg nim men ten VAT LIEU VA PHVONG PHAP Chting, hia chit vi mai truivng, dyng cy vi thiet bi Ching nam men P pastoris X33::pdgf-b duqc A cung cap bai Be mon CNSH Phan tin va MN truing, Trtkmg Dai hoc Khoa hoc tu nhien, DHQG-HCM Chung mang 16 ban gen bieu hien protein rhPDGF-BB duqc too bang each chin plasmid pPICZaA mang gen pdgf-b vao be gen, (fang thoi di duqc kiem tra Ming hien hien, cho ditty mirc de tiet protein ngoai lai vii se luqng km M6i truing nu8i cay vi sinh gem: YPD (1% cao nim men, 2% peptone, 2% glucose, sir dyng be sung 100 mL dung djch glucose 20%), YPD-zeocine 100 (YPD be sung 2% agar va khang sinh Zeocin den Ming de cuei 100 ug/mL), BMGY pH4 (1% cao nim men, 2% peptone, 1,34% YNB, 10% CPB 10X, sir dung be sung biotin 500X den neng de cuei la X va glycerol 10% den neng de cuei li 1%), BMMY (BMGY duce thay the glycerol bang methanol (MeOH) c6 neng d6 cuoi la 0,5%) (Invitrogen, 2014) Dung cu va thiet bi: he thong I9c tiep tuyen vii cat lac 10 kDa, he thong tinh che AKTA FPLC, cat sac ki trao doi cation Hitrap'SP FF mL va cat sic ki ki ntroc Phenyl Sapharose mL duqc cung cap bai hang GE Health care Cam Ong bleu hien rhPDGF-BB ti• chiing X33::pdgf-b 'Quin lac ten dia men truing YPD-zeocin 100 duqc hoot h6a so cep moi twang YPD, sau da 822 hoot hie dui cap m8i twang BMGY pH4 bang citch nuoi cay lie a 28°C Yeti teic de 200 Ong/phut cho tai OD600 dat - (khoang 18-24 gi6) Li tarn thu sinh khei to bao, tien hanh cam irng bieu hien 300 mL moi twang BMMY pH4 v&i met de OD600 ban du bang 1, dieu kien nhiet de la 28°C, tec de lic la 180 vang/phut vi be sung Me0H ding d6 cu6i 0,5% cir sau mei 24 gib Thai gian cam (mg la 120 gib Khao sit quy trinh thu nh#n rhPDGF-BB Djch nu8i cay sau 120 gia dirge li tam loci xac to bao Djch not chira rhPDGF-BB duqc khao sit thu nhon bang hai phuang phap: tint yeti (NH4)2SO4 bao haa a clic neng de 40%, 50%, 60% va 70%; va 19c tiep tuyen a hai tee de I9c 125 Yong/phut vi 150 vang/phin Trong quy trinh tira, (NH4)2SO4 duce them vao 100 mL djch mau va khuiy deu a 11°C gi6 Thu tila bang cach li tam 5000 Ong 20 phut, matt tira sau d duqc haa loi vOi 50 mL dem Tris-HCI 20 mM pH7,5 Mau Ma haa tan va mau djch not sau li tam duqc dem di phin tich bang dien di SDS-PAGE dung thai djnh luqng protein king bang phuang phap d o Bradford vi djnh luqng tuang d6 i protein muc tieu bang phan mem ImageJ de tinh Win hieu suit Trong quy trinh 19c, 200 mL djch mau dirge 19c qua he thong lac tiep tuyen QuixStandTM (GE Healthcare) yeti cat l9c 10 kDa Sau gia, djch cat vi djch ngoai cat duqc thu nhin de dien di kiem tra vi djnh !wing tuang tu nhu a quy trinh Ma Khaio sit quy trinh tinh chi bang sic Id trao dei cation va sac ki Id ntrac Trong quy trinh tinh che bang sic ki trao doi cation, cat HitrapTm SP FF mL (GE Heathcare) duqc sir dung Mau sau 19c tiep tuyen duqc dal sang dem acetate 20 mM pH4 (dem A) bing phuang phap thim tich Quy trinh tinh che tuan tu nhu sau: can Wing cat yeti dem A, nap 20 mL mau, ria cat bang 10 mL dem A, Ii giai protein myc tieu bang phuang phip dung li gradient tir — 100% vii dung djch li giai (dem B) la dung djch dem A be sung NaC1 M Tee de clang mL/phirt va phin (loan Ii giai co the tich mL Citc phin down protein thu sau li giai duqc phin tich bang dial di SDS-PAGE va djnh Itrqng de kiem tra da tinh sach ding nhu tinh tom hieu suit tinh che Doi vii quy trinh tinh che bang phuang phip sic ki ki nuac, cat Phenyl Sepharose mL (GE Healthcare) duqc sir dung Quy trinh duqc thuc hien ttrcrng ter nhu phuang phip sic ki trao dai cation, Top chi Cong nghe Sinh hpc 13(3): 821-830, 2015 46 am A li dung djch (NH4)2SO4 1,4 Mem phosphate 50 mM pH7 va dem B la mac cat miliQ Khio sit mOt s6 yeu to inh hurting qua trinh tinh che teen cat sic Id trao 46i cation Khho sift pH cua dung djch can bang pH cua dung dich can bang duerc khio sit ba gifi tri 4, vi 8; 46 pH4 sir dung am acetate 20 mM (dem Al), pH6 khio sit a hai dam: acetate 20 mM (dem A2) vi phosphate 20 mM (dem A3); vi pH8 sir dung dem Tris-HC1 20 mM (dem A4) mai gia tri pH khio sit, phin dung djch kh6i cat bulk nap mL duqc thu nh#n vi phan tich bang dien di SDS-PAGE de dinh gifi inh Wong dm pH dung djch can bang len ning gin cat cua protein muc tieu D6ng th6i cic phan Joan protein thu sau li giii cling duqc dien di vi dinh luqng de tinh town hieu suit tinh che Sau qui trinh khio sat, gii trj pH phi hqp nhit se duqc sir dung cac quy trinh khio sit tiep theo Kith() sot dung djch li giai Sau bim vio chit trao i ion vi duerc giv lai ten cat, hPDGF-BB c6 the dtrqc li giii khai,cat bang each tang pH, tang 19c ion hoc ket hop &mg th&i ca tang pH vi tang lure ion De so sinh giira cic each li giii nay, qui trinh li giii Querc tien hinh vii ba dung djch li giii khic d#c trung cho mai cich hi: dung dich NaCI I M/dem acetate 20 mM pH4 (am BI), dung djch Tris-HC1 20 mM pH7,5 (dem B2) vi dung djch NaCI M/Tris-HCI 20 mM pH7,5 (dem B3) Cfic phan an protein thu sau li giii Quqc phan tich de tinh town hien suit cola phuang phip tinh che KAT QUA VA THAO LUAN Khio sit quy trinh thu nh#n rhPDGF-BB Thu nhtln bang pherang pheip tha mIu wfri (NH4) 2SO4 bao hoa Ching toi tien hinh khio sit tim 100 mL mau o cac mang CIO 40%, 50%, 60% vi 70% (N114)2SO4 bao nhim 19a ch9n nong d6 mu& 46 protein time tieu Quqc tits toi da Hinh IA cho thiy cac mau tim (gieng 2) au co vach protein mix tieu (30 kDa) tuang img vii tau truac tim (gieng I) Cic mau dich not (gieng 3) hau nhu khong xuit hien vach protein muc tieu Nhu v3y, protein muc tieu deu eltrerc dm ci b6n nOng 46 (NH4)2SO4 bio Ket qui tinh town hieu suit thu h6i vi de tinh sach (ti le tuong doi cia rhPDGF-BB so.v6i protein dm (Bing 1A) cho thay hieu suit tong) dm cic thu nh#n ttia cic cac ming 40 (NH4)2SO4 bao h6a 50%, 60% vi 70% gin nhu tuong duong nhau, dat 78,1%, 81,9% vi 82,0% Wang irng; vi cao hem khoing 1,15 Ian so vii dm 40% (NH4)2SO4 bao Trong d 6, de tinh sach tuong i cua cac mL dat cao nhit (54 - 55%) tua n6ng deo bao has 60% vi 70% Nhu 4y, ming a 60% vi 70% (NH4)2504 bao boa, protein muc tieu duce tii da vii hieu suit thu nhon vi 46 tinh sach tuong throng Xet ve khia canh chi phi sin xuit, ching Col de nghi tieing 46 60% (NH4)2SO4 bao haa la nang mau tii uu de thu nh#n rhPDGF-BB tir dich protein tong sau nuoi cAy Thu nkIn bang phuang phap !pc dip tuyin Tic a 19c the inh huerng den hieu suit thu hill sin phim sau 19c De cheat duerc toe I9c phi hqp, rut ngin th6i gian I9c nhung van bio dim duqc MO suit thu h6i, ching toi tien hinh khio sit hai tOc a 1QC li 125 vonyphin va 150 yang/phut ap dung cho cat 19c tiep tuyen vii ming 19c 10 kDa IC& qui (lien di SDS-PAGE (Hinh [B) cho thiy ci‘ hai tOc I9c, dung dich cat (gieng vi 4) deu c6 vach protein muc tieu Mang duang protein mL truic I9c (gieng 1), dung dich khai cat (gieng vi 5) kh6ng xuit hien vach protein nio Dieu 46 ching to protein muc tieu da ducrc gitt lai vi nhung protein tap yell kich thuac < 10 kDa bj day khoi ming (cic protein Bong thiy tren bin dien di) De dinh gii inh htrang dm tic 40 19c len viec thu nhiin sinyham muc tieu, ching toi tien hinh so sinh hieu suit thu nhOn sin pham sau I9c hai tOc 46 Bing 1B cho thiy hieu suit thu nhan hai tOc a 19c la ngang (khoing 64%) Tuy nhien, lucmg protein muc tieu bi mit Qi.sau 19c nhieu (khoing 36%), Qieu c6 the protein hi cic protease ngoai bio dich len men phan giii qui trinh 19c Ket qui thu dtr9c sau khio sit co mL bang phuang phip tita (NH.4)2SO4 biio haa va I9c tiep tuyen li tien de khio sat ip dung hai phuang phip thu nh rt o quy ma sin xuat Ian hem M#c di hieu suit thu nho'n cua phuang phap I9c tiep tuyen (63,23%) thap han 1,3 Ian so yeti hieu suit 823 Vu Kha Thanh Thanh et al thu nhan cua phuang phap tin (81,90%), chi:mg Col phuang phap thi,rc hign dun gian va tit ki01 sir dvng phuang phap de thu nhAn mau cho cac thZri gian han (doi vai th6" tich mau la 200 mL, qua buck MIA() sat quy trinh tinh the tie)) theo, trinh lye chi mat gia tua mat 12 gia) 40% 50% 70% 60% A LBW 125 150 vang/phait rvenygwt 94 las 67 43 B 30 20 14.4 Hinh Kha nang co protein (A) tua mat.' a cac nang dO 40%, 50%, 60%, 70% (NH4)2SO4 bao haa (1: mat, ban deu tri.rac tua (V=100 mL); 2: mAu taa sau Ii tam duqc hoa vai dam Tris-HCI 20 mM pH7,5 (V=50 mL); 3: mat, dich noi sau Ia tam) va (B) iQC ti6p tuyen a cac tic dO loc khac (1: meu ban deu trutfc loc (V=200 mL); va 4: dung dich gift cOt sau qua trinh loc (V=100 mL); va 5: dung dich khdi cOt sau qua trinh lqc (V=100 mL) Bang Hieu suet thu nhan va dO tinh sach caa hPDGF-BB (A) tua b cac n6ng dO (NH4)2SO4 baokhac va (B) lqc tip tuyen cac tic d !pc khac Mau A B Tnr6c tua Sau tua 40% (NH4 2SO4 bao hem Sau tua 50% (NH4)2SO4 bao Ma Sau tua 60% (NH4)2SO4 bao hoa Sau tua 70% (NH4)2SO4 bao hoa Trtyac Ipc tiip HiOu suit thu nhOn (%) 48,00 Lircmg hPDGF-BB twang dOi (mg) 8,33 5,81 14 78 44 00 6,51 78,10 0,25 12,63 54.00 6,82 81,90 50 0,25 12,42 55,00 6,83 82,00 200 0,19 37,74 50,00 18,87 100,00 100 0,22 22,10 54,00 11,93 63,23 100 0,22 21,77 56,00 12,19 64,62 Lircmg protein tong (mg) Ti le twang dei hPDGFBB/protein tong (%) 0,17 17.00 49,00 0,24 12 10 50 0,3 50 The tich (mL) Nang de protein (mg/mL) 100 50 100,00 69,71 tuy6n Dich cOt (125 vOng/phat) Dich cOt (150 vong/phat) 824 Top chi Cong nghe Sinh hoc 13(3): 821-830, 2015 Khao sat quy trinh tinh cite hang sic id trao d61 cation va sac ki kj nu& Siic ki trao al cation Protein muc tieu duqc bieu hien mai truing nuoi city c6 pH4, vi vay tien hanh tinh che a pH D Ong th&i rhPDGF-BB c6 diem ding di en (pI) bang 9,8 (Antoniades et al., 1979) nen pH < 9,8, protein tich dien ducmg, thich hqp tinh che bang phuang phap sac ki trao d6i cation Khi kinio sat tinh che bing phtrcmg phap dung Ii gradient tren cat trao deli cation SP FF mL, so vii protein ding ban din, phAn lem cac protein tap kitting mong muen den di khoi cat buac nap mAu va buac rim cat sau nap mAu (Hinh 2A, gieng va 3) Nhu viy, bang cach sir dung sac ki trao deli cation c6 the loai dirge nhieu protein tap c6 Ngoai ra, a cac gieng va 6, ket qua cho thay c6 mat luqng protein muc tieu duqc dung li a khoang 60% dung dich Ii giai, nhien lucmg khong ding ke va a nheng phan doan protein muc tieu can lin vii cac protein tap khac nen chung tai chi thu nhin phan doan tinh sach tir 76 — 93% dung djch Ii giai, nhimg phan doan hiu nhu kitting c6 tap Ket qua a bang 2A cho thiy, neu chi thu hal cac phin dcgm et gieng (Hinh 2A) thi MO suit tinh che san phim d at khoang 15% va d a tinh sach cua protein thu duqc la 99% Day la buac khoi diu hieu qua cho flitting thi nghiem sau Sitc ki kE nu& LMW 94 kDa 67 Neu chqn phucmg phap tita a buac thu nhin thi (N114)2SO4 ding la tic nhan lam tang tinh kj ntrac cho protein Do de, rhPDGF-BB cling c6 the duqc tinh che bang phuang phap sac ki ki, ntrerc Ket qua dien di SDS-PAGE san pham tinh che bang sac ki kj nuac tren cOt Phenyl (Hinh 2B) cho thay, et gieng khOng c6 stir xuat hien dm vach protein muc tieu, nghia la protein muc tieu da barn het len cat qua trinh mAu Nhang protein tap tai c6 luc tuong tic yeu vii chat nen sac ki, bj dung Ii tang dan dung djch Ii giai (gieng 3) d On khoang 80% Protein muc tieu twang tic mph vii cat va chi ducrc dung li n8ng da tir 82 - 100% dung djch li giai (gieng 4) Protein rhPDGF-BB thu dtrqc sau tinh che bang cat Phenyl Sepharose mL c6 d a tinh sach 93% va hieu suit thu hai Qat 12,05% (Bang 2B) Tuy nhien ap dung phuang phap sac ki kj ntrac tren quy m8 san xuAt Ion thi nen can nhic vi se ton rat nhieu chi phi cho h6a chit de Qua dich protein matt ban &Au ve cling vii dung djch dem can bang cat So sanh giita phuang phap sic ki trao dal cation va sac ki kj nuerc ye ket qua tinh che, to thAy hieu qua sir dung phuang phap sac ki trao doi cation la tot han vii da tinh such 99% va hieu suit tinh che 15% Do do, de chi sir dung mat cat sac ki ma hieu suit tinh che yin d at yeu cAu (>95%), buac d Au chung tai quyet d inh chqn phucrng phap sac ki trao dOi cation de khao sat cac thi nghiem tiep theo cua quy trinh UAW 94101 67 43 43 30 30 20 20 14.4 14,4 A B Hinh Ket qua tinh che rhPDGF-BB bang phuang phap dung li theo ding gradient (A) tren cOt Hitrapw SP FF 5mL (1: m5u tru:ac tinh che; 2: dich khoi cOt a buac nap m5u: 3: dich khOi cet a buac rira; 4: phan doan lir - 55% dem 13; 5: phan doan hj: 56 - 69% dem B: 6: phan doan Ur 70 - 75% dem B; 7: phan doan to 76 - 93% dem B: 8: phan doan tir 94 - 100% dem B) va (B) tren cOt Phenyl Sepharose (1: m5u triyac tinh che; 2: dung dich khoi cOt buttc nap m5u; 3: phan doan protein duvc dung li tiy - 81% dem B 4: phan doan protein duqc dung li Ur: 82- 100% dem B) 815 Vi Kha Thanh Thanh et al Bang HiOu suit tinh che rhPDGF-BB b0ng phtrang phap dung li gradient (A) tren cOt HitrapTM SP FF mL va (B) ten cOt Phenyl Sepharose mL Mau A B The tich (mL) N6ng d6 protein (pg/mL) Luang Ti 10 twang d6i hPDGF-BB/ protein tong (%) Luang hPDGF-BB twang dOi (1.19) Hieu protein tong (1-19) Trubc tinh che 20 89,23 1784.60 40.00 713,84 14.75 Phan down china hPDGFBB tinh sach 18 5,91 106.38 99,00 105,32 Trutfc tinh che 20 85,57 1711.4 45,00 770,13 Phan down china hPDGFBB tinh such 19 5,25 99,75 93,00 92,77 Khio sit dieu kiln him ctit va li giai guy trinh tinh che bang sac ki trao dui cation Hieu suit tinh che va de tinh sach cia protein mvc tieu thu duqc thu6ng chju inh huang curt nhieu yeu to qui trinh tinh ,che Vi vay sau da chgn duqc phuong phip sac ki phi hqp can tien hanh khao sit dung djch can bang va dung djch li giai nham mvc djch tang hieu suit tinh the ding nhu de tinh sach cia sin pham Khio sit pH cis dung djch can bing pH la met th6ng so quan tong dm dung djch can bang, nd anh hudng d in nang gin cia protein mvc tieu ding nhu cac protein tap tren cot Cic dem can bang khao sit duce ki hieu nhu sau: Al (dem acetate 20 mM pH4), A2 (dem acetate 20 mM pH6), A3 (dem phosphate 20 mM pH6) va A4 (dem Tris-HC1 20 mM pH8) Ket qua dien di (Hinh 3A) cho thAy pH khic co inh htrang re ret len rang bim cot cia rhPDGF-BB cac dem A2, A3 va A4 cd met hrong protein mvc tieu kitting bim vio chit trao doi cation tren cOt, dem Al hat' nhtr protein mvc flit' barn het lin cot Nhu vay, or dem Al mkt barn OA tot nhAt C6 the pH4 la gii trj xa yeti diem dang di en (p1=9,8) cua rhPDGF-BB nhAt nen nang gin v6i chit trao del cation tren cot sic ki la cao nhit Tuy nhien, d e kiem tra pH lin can pH4 co the giip gia tang nang bam cot cia rhPDGF-BB hay 826 swat tinh che (%) 12,05 kitting, citing Vol tiep tic khao sit them hai gii trj la pH3 va pH5 Ba dem can bang: A5 (dem Glycin-HC1 20 mM, pH3), dem Al (dem acetate 20 mM, pH4) va A6 (dem acetate 20 mM, pH5) duqc so sinh cuing tu nhu ten Ket qui dien di (Hinh 3B) cho thiy dem A5 vi A6 co met ltiong rhPDGF-BB kh6ng bam vao chit trao dOi cation cot va di ngoai cing v6i cac protein tap Nhu voy, pH4 maw van bim cot tot nhAt Tnr6ng hqp pH3 (dem A5), protein mvc tieu bim kh6ng tot, nguyen nhan c6 the pH qua thip, protein bj thay dei cAu tric dart den !tang gan v6i chit nen dm cot sac ki ding thay doi Ngoai ra, Wong va phan protein barn teen cet cing c6 the duqc danh gii thong qua protein thu nhan duqc sau qui trinh li giii De cd the so sinh gifra cac pH khao sit, qui trinh li giai dtrqc Wen hinh vOi cing phucmg phip vi dung djch nhu da dtrqc m6 Hinh 3C (ket qui djnh luqng kitting trinh bay chi tiet) cho thiy de tinh sach dm protein rhPDGF-BB sau tinh chi khio sit cac pH khic mkt dem can bang li xip xi (deu > 95%) vi hieu suet tinh che dot cao nhat (khoing 16%) sir dung dem can bang acetate 20 mM pH4 Nhu vay de tinh sach va hieu suet tinh che sir ding dem can bang acetate 20 mM pH4 dot cao nhAt (99% va 16%) Do do, citing toi chqn pH4 la gii trj pH ma mau barn cot tot nhAt vi sir ding dung djch can bang cho cac thi nghiem khao sat tiep theo Top chi Cong nghe Sinh Imc 13(3): 821-830, 2015 LMW LIM S AS Al A6 A2 Al S 94 kDa 14 67 67 43 43 30 30 20 20 14,4 14,4 A B 99 100 97 -98 96 94 92 , 90 Dem Dem Dem A4 AS AS Sim Dim Dim A3 Al A2 ■ (%) 3es 444 co 97 Hiitt suit tinh chi (%) tinh sach (%) C Hinh Ket qua khao sat dieu kien barn cot veri cac dem can bang khac A, B: Ket qua dien di SDS-PAGE minh hoa kha nang barn cot cCia rhPDGF-BB (A) & cac dem can bang: Al, A2, A3 A4 va (B) & cac dem A5, Al, A6 (S: m5u taxac qua cat) C: flo tinh sach va hieu suAt tinh ch6 protein str dung cac dem can bang khac Khan sat dung djch li giai Cac dung djch li giai cluqc khao sat bao Om: Om B1 - dung djch NaCI M/dem acetate 20 mM pH4 (tang 1uc ion), dem B2 - dung djch Tris-HCI 20 mM pH7,5 (tang pH) va dem B3 - dung dich NaCI I M/Tris-HCI 20 mM pH7,5 (tang luc ion va pH) Do ding sir dung chung mOt dem can bing by& can bang cOt nen !yang protein barn cOt IA nhu o ca ba thi nfhiem khao sat, vi the hinh kh6ng trinh bay gieng dung djch khoi cOt buck nap mAu Nhin chung, protein muc tieu va gip &vac li giai ar cac phan down khac Tuy nhien dem B2 khong thu &Km phan down tinh such (6 gieng 100%, ngoai vach protein muc tieu co lin ntsiieu vach protein tap khac) chiing to viec giai bang cach tang pH kh8ng hieu qua twang hop Do de, chimg toi chi tien hinh so sanh hieu suit thu hoi doi vOl quy trinh sir dung dem li giai NaCI M/dem acetate 20 mM pH4 va NaCI M/Tris-HCI 20 mM pH7,5 Ket qua djnh Wang cho thiy a ca hai quy trinh khao sat deu cho dO tinh such cua rhPDGF-BB IA twang &rang (99%) Tuy nhien, hieu suit tinh the san phim sir dung dem li giai NaCI M/Tris-HC1 20 mM pH7,5 (dot khoang 22%) cao khoang 1,4 lin so vai sir dung dem NaCI M/dem acetate 20 mMs pH4 (dot khoang 16%) Do 46, chimg t6i by& dau ket luon chon sir dung dem NaCI M/Tris-HCI 20 mM pH7,5 de li giai protein muc tieu bing phuang phap dung li theo gradient quy trinh tinh the sir dung cOt SP FF mL 827 va Kha Thanh Thanh et al LMW S 56-75% 76-93% S 53-99% 100% S 54-66% 67-87% 94 kDa 67 43 30 20 14,4 B1 B2 B3 Hinh Ket qua tinh che a cac dem li giai: B1, B2 va B3 (S: mau truck tinh che) LMW LMW 94 kDa 67 43 30 20 14.4 A B Hinh Ket qua dien di SDS-PAGE (A) va lai Western - blot (B) vii khang the khang rhPDGF-BB mau tinh che bang phtrang phap dung li theo gradient tren cet SP FF 5mL (1: mau trip:5'c tinh che: 2: mau tinh sach thu ductc a phan down dung li 67 - 87% dem B) Nhu vOy, chAng toi d A thu nhAn ding rhPDGF-BB tai to hqp co de tinh such cao 99% (len ham 95% IA dO tinh such toi thieu cen thiet cho cac sin phim sir ding cho mic dich y sinh) vai hieu suit tinh che khoing 22% D busk d Au d ant) gia rhPDGF-BB thu dtrqc sau qua trinh tinh che, chAng toi tien Matt dien di SDS-PAGE vi lai Western — blot kiem tra Ket qui lai Western - blot vai khang ,the dAc hieu khang rhPDGF-BB (Hinh 5) cho thay phin doon dung Ii 67 - 87% dung dich NaCI I M/Tris-HCI 20 mM pH7,5 cho ket qua duang tinh twang Ong vOi voch tren bin dien di SDS-PAGE Do do, ching toi 828 khAng djnh protein thu nhAn dtrqc sau qui trinh tinh che chinh IA rhPDGF-BB mic tieu Hoot tinh sinh hoc cita protein sau tinh che se duqc tiep tic kiem tra cac nghien eau sau KET LUAN Trong btrac thu nhon protein ban dAti ,trtrac tinh che, protein mic tieu co the thu nhAn toi da bang phtrang phap tint 60% ammonium sulfate bio hos hoAc phuang phi!) lqc tiep tuyen yeti toc d 125 Ong/phut Day IA den de cho viec khao sat phuang phi!) thu nhAn tren quy m6 km Top chi Cong nghe Sinh hoc 13(3): 821-830, 2015 Bulk au d a xay dung du qc quy trinh tinh che cho a tinh sach 99% va hieu suit (1 at khoang 22% bang phucmg phap sac ki trao deti cation tren Ott SP FF mL Trong quy trinh tinh che, dung djch can bang duqc chonla dem acetate 20 mM, pH4 de mlu c6 the barn cat tot nhat va dung djch li iiai dtroc chip la NaC1 M/Tris-HCI 20 mM pH7,5 de dot hieu suet tinh che cao nhit Neu luong protein kh8ng bj mat qua trinh bao quart thi hieu suit thu hoi cita ca qua trinh va lucmg protein mix tieu tuang d of thu dugc tir 200 mL djch len men u ac tinh thu duqc la 13,25% va 2,5 mg nen sir dung phucmg phap loc tiep tuyen buerc thu nhan protein; la 18% va mg neu sir dung phucmg phip tits d e thu nhan protein ,Protein tinh soch.sau tinh che duqc sir dung de tien harsh thin tren to bao de danh gia hoot tinh sinh hoc Lai cam an: nghien ciiu duqc thgc hien tit nguem kink phi ciia de tai nghien ctiu khoa hoc cap Sde Khoa hoc va Cong nghe Thanh Ho Chi Minh "Nghien cuu tgo yeu 10 king truong tai to hqp 1u lieu cau (Platelet-derived growth factor-PDGF) nheim dieu tri kit ban chan d thao du emg" (so` hqp dung: 219/2013/HDDH-SKHCN) TAI LIEU THAM KHAO Antoniades NH, Scher CD, Stiles CD (1979) Purification of human platelet-derived growth factor J Cell Biol 76: 1809-1813 Blatti SP, Foster DN, Ranganathan G, Moses HL, Getz MJ (1988) Induction of fibronectin gene transcription and mRNA is a primary response to growth-factor stimulation of AKR-2B cells Proc Nail Acad Sci USA 85: 1119-1123 Choi JH, Kim S, Sapkota K, Park SE, Kim SJ (2011) Expression and production of therapeutic recombinant human platelet-derived growth factor-BB in Pleurotus eryngii Appl Biochem Biotechnol 165: 611-623 Darby RAJ, Cartwright SP, Dilworth MV, Bill RM (2012) Which yeast species shall I choose? Saccharomyces cerevisiae versus Pichia pastoris (review) Springer 866: 11-23 Deepa K, Rodionov RN, Weiss N, Parani M (2013) Transgenic expression and functional characterization of human platelet derived growth factor BB (hPDGF-BB) in tobacco (Nicotiana tabacum L.) App! Biochem Biotechnol 171: 1390-1404 Embil JM, Nagai MK (2002) Becaplermin: Recombinant platelet derived growth factor, a new treatment for healing diabetic foot ulcers Expert Opin Biol Ther 2: 211-218 Glenn FP, Thomas AM, Bruce WA, Thomas FD, Arlen T (1991) Role of platelet-derived growth factor in wound healing J Cell Biol 45: 319-326 Heldin CH, Westermark B (1999) Mechanism of action and in vivo role of platelet-derived growth factor Physiol Rev 79: 1284-1301 lnvitrogen (2014) Pichia Expression Kit User Guide Life Technologies Corporation 1-97 Karumuri NN, Gangireddy SR, Narala VR, Majee SS, Gunwar S, Reddy RC (2007) Simple, rapid, high-purity preparation of recombinant human platelet-derived growth factor-BB Biotechnol Lett 29: 1333-1339 Lepistoe 1, Laato M, Niinikoski J, Lundberg C (1992) Effects of homodimeric isoforms of platelet-derived growth factor (PDGF-AA and PDGF-BB) on wound healing in rat J Surg Res 53: 596-601 Ricardo HA, Hagopm MK, Jorge EC (2006) Biology of platelet-derived growth factor and its involvement in disease Mayo Clin Proc 81: 1241-1257 Schonherr E, Jarveliinen HT, Sandell LJ, Wight TN (1991) Effects of platelet-derived growth factor and transforming growth factor-beta on the synthesis of a large versican-like chondroitin sulfate proteoglycan by arterial smooth muscle cells Biol chem 266: 17640-17647 Wang Y, Xue L, Li Y, Zhu Y, Yang B, Wang X (2009) High-level secretory production of recombinant human platelet-derived growth factor-BB by Saccharomyces cerevisiae under the non-selective conditions Appl Biochem Microbiol 45: 156-161 829 VO Kha Thanh Thanh et a! PURIFICATION OF RECOMBINANT HUMAN PLATELET — DERIVED GROWTH FACTOR-BB (rhPDGF-BB) Vu Kha Thanh Thanh, Ngo Thi Huyen Trang, Nguyen Tri Nhanc=3, Tran Linh Thuoc, Dang Thi Phuong Thao University of Science, VNU HCM City SUMMARY Recombinant human platelet - derived growth factor-BB (rhPDGF-BB) has been widely applied for various therapeutic purposes, especially in the treatment of diabetic foot ulcer The expression systems of Escherichia coli and Saccharomyces cerevisiae have been used for the production of rhPDGF-BB However, Pichia pastoris has attracted considerable interest in recent years because of its high volumetric productivity Vuong Cat Khanh et al (2014) have succeeded in constructing of a recombinant P pastoris clone, which carries multi-copy of hPDGF-BB coding gene In this study, we performed the selection of suitable techniques for the initial concentration and the purification of rhPDGF-BB from the culture of the multi-copy P pastoris clone The yields of the initial concentration processes of rhPDGF-BB by amonium sulfate precipitation and tangential filter with 10 kDa membranes were 82% and 63%, respectively Purifying the target protein by cation exchange chromatography gave higher yield and purity in compare with that by hydrophobic interaction chromatography In the refining process of cation exchange chromatography, the binding buffer and the elution buffer was chosen as 20 mM acetate buffer (pH4.0) and I M NaCl/20 mM Tris-HC1 buffer (pH7.5), respectively, to achieve the highest performance with approximately 99% of purity and 22% of yield Keywords: Amonium sulfate precipitation, cation exchange chromatography, hydrophobic interaction chromatography, rhPDGF-BB, tangential filter 4° Author for correspondence: Tel: +84-8-38307079; E-mail: ntrnhanahcmus.edu.vn 830 ... to hqp mang 16 ban gen ma h6a protein hPDGFBB Trong nghien eau nay, ching Col tiep tic tien hanh khao sit quy trinh thu nhan va tinh che rhPDGF- BB a quy m6 ph6ng thi nghiem sir dyng chimg nim... de kiem tra da tinh sach ding nhu tinh tom hieu suit tinh che Doi vii quy trinh tinh che bang phuang phip sic ki ki nuac, cat Phenyl Sepharose mL (GE Healthcare) duqc sir dung Quy trinh duqc... trip:5'c tinh che: 2: mau tinh sach thu ductc a phan down dung li 67 - 87% dem B) Nhu vOy, chAng toi d A thu nhAn ding rhPDGF- BB tai to hqp co de tinh such cao 99% (len ham 95% IA dO tinh such
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