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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 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 15 thOng 09 min, 2014, nkin dang 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 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 tag len Al' bieu hihn B subtilis Ket qua bleu hihn protein tai to hqp Ngoai d6c dihm cho they ss,r Neu hihn cua GFP mang mOt promoter manh, hai plasmid subtilis giam rnpnh dung hop voi His-tag can dugc tich hqp them duoi tinh the His-tag tpi dhu N Day se la thong tin quan trverc vOng MCS (Multi Cloning Site) giOp Binh huong sO' dyng cOng nhu' thief ke pHT253 (Pgrac100-8xHis-MCS) va sau wing thi nghihm bleu hihn tinh the protein MCS 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 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 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 met chisng this kha hty dan 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 Trang Science & Technology Development, Vol 17, 1(1.14-2014 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 &roc dung hop them duel tinh sach 8xHis-tag vao truetc va sau vung MCS, tao 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 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) dung hop vac) du N ho5c C B subtilis, twang hop 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 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 bulk dong hoa va B subtilis 1012 &roc ding de danh gia inh htremg cua 8xHis-tag len so bieu hien cita GFP thi nghiem Ccic plasmid dune sir dung bao gem pHT254 cilia promoter Pgrac100 vi gen 8xHis am sau vung MCS; plasmid pHT1169 di dtroc tao 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 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 enzyme BrunH1 va And' va not vao pHT254 cling da dupe xu ly cling loai enzyme tao vector pHT1170 (Hinh 1B) Qua trinh sang loc dupe thoc hien 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 ruing de IPTG (0 mM; 0,001 rnM, 0,01 mM; 0,1 mM; 0,5 mM va mM) U a 37°C 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 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 lan, m8i Ian tren khuan lac khac Cam sing bieu hien GFP Ching B subtilis 1012 china cac plasmid pHTI169, pHT1170 dune nal cay 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 — vii cac ming de mM; 0,001 mM; 0,01 mM; 0,1 mM; 0,5 mM vi mM Thu mau 0, 2, gi6 sau cam ling ly tam 6000 yang/phut 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 gier sau cam ung vai mau chimp pHT01 Thi nghiem &roc lap lai lan tren khan lac khac Mau sau 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 60 ul PBS (Phosphate Buffer Saline), them lysozyme dot neng de cuei la I ug/p.1, 6 37°C phfit, sau 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 ly se &cm u 95°C phit, ly tam 10.000 vang/phtit 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 bed acid amin Serine65-Tyrosine66-Glycine67 tai tam host Ong dm churl polypeptide Qua trinh ding yang la met chwii cac phan img hydro vi oxy hoi, tao 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 30 plait Dich chiet to bao sau ly tam &Km 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 lan tren khan lac khac KET QUA Da dimh gia inh hiring dm His-tag len 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 phan Vat lieu phuang phip Trong nghien cfru nay, plasmid pHT1169 (Hinh 1A) diroc si chmg thu met del cluing cO cau trite tucmg to pHT1170 (Hinh 1B), nhung 8xHis7tag se chrac gan vat) dau N ctla GFP 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 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 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 dang dung hop 8xHis-GFP va GFP-8xHis theo thin to Trang Science Technology Development, Vol 17, No.T4-2014 Danh gia anh huiing caa SxHis-tag 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 chArn khutin lac tren cac &a thach chaa khang sinh va nong de IPTG khac Ket qua sau a 16 gia qua dem 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 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 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 se &roc kiem chain bang cac tht nghiem tiep theo B subtilis 1012 Gray value 0,001 0,01 0,1 0,5 mM pHT01 IPTG B 120 100 80 pHT1169 60 40 pHT1170 20 pHT01 pHT1169 pHT1170 Huth 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 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 dieu kien nhu dA me too phan Vat lieu phtrong phap Mau duqc thu a cac th6i diem 0, 2, gier sau 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 dAu N va C dime the hien a Hinh So skill vii ching Am pHT01, chin B sztbtilislpHT1170 (Hinh 3B) cho kha nAng bieu hien GFP virgt mix nhang nong de) IPTG cao Trang (0,1, 0,5 WI mM) Ira tang dtin theo th&i gian cam (mg Tuy nhicn, vach protein muc tieu 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 dau C (pHT1170) Dieu hoax' toan khop vii thi nghiem khito sat hoat tinh tren &a thach Su anh huang 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 gia cam img Doi yen pHT1169, muc bleu hien kha thap clan den su che'nh lech giUa cac nghiem thirc la khong ding ke Ket qua 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 B subtilis Trong khi, His-tag a dttu C cho mire de bleu hien kha tot, cao gAp nhieu ltin so vai twang hop 14i Thio luin Huth Kat qua khito sat Mau hien cita GFP bang phuong phap dien di SDS-PAGE chang B subtilislpHT1169 (A) va B subtilis/pHT1170 (B) M, Thang protein; (-), pHT01 MAu thu 60, 2, gia sau cam img vai IPTG b cac nAng khac Dinh gii anh hiring elm 8xHis-tag mai truing long sir dyng phirong phip host tinh teen dia 38-1 gieng Phuang phap cueing de huynh quang co de chinh xac cao han phixang phap tren Wing de GFP cang cao thi gia tri huynh quang cang 16n va nguoc lai, qua c6 the dinh gia mile dO bieu hien ciut GFP nhim dua &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 So sanh ctrang de phat huynh quang cint 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 se gay met su Un tic, lam cbAm qua Wirth dich ma neu nhu tinh trang thieu but t-RNA mang histidine dien Hoc ea kha nang, dau N cita GFP c6 vai trO quan trong viec bieu hien cling nhu quyet dinh hoat tinh sau dich N se anh ma, duel poly-histidine xuat hien huerng sden qua trinh gap cuOn, lam giam hog tinh ciia protein [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 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 protein chi thi GFP va BgaB Nhung day khong phai la thOng tin chung ma tity thuec vao protein muc lieu Trang Science & Technology Development, Vol 17, No.T4-2014 B subtilis 1012 / pHT1169 Don vj hoat tinh 1012 / pHT1170 2000 • mM 80 B subtilis Dom vi hot tinh M0,001 1B00 • 0,01 200 • 0,1 40 ■ 0,5 BOO • mM 400 gib gib Ws o gib gib gib ThM 1-finh 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 then them gen 8xHis vao trunk gen gjp; do, dOi yeti plasmid pHT1170 (cling chira promoter Pgrac100, dudi His va gen gfp), nhung luc gen 8xHis /lam sau gen muc lieu thi kha nang bieu hien GFP cao hon gap nhieu lam Dieu 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 chien ltroc sir dung pHT253 va pHT254 Nghien ctru &tor tat tro bed 121.12 phat tplin khoa hoc va tong nghe au& gia (NAFOSTED) 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 Trang 10 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: http://www.mobitec.com/cms/prod 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) Trang 11 ... 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. .. pHT1170 (Hinh 1B) Qua trinh sang loc dupe thoc hien E colt Omni MAX vdi mei ON1249(5'CGTTTCCACCGGAATTAGCTTG-3'), ONI359 (5'ACGTACGATCTTTCAGCCGACTC-3'), plasmid dung dupe bien nap vao B subtilis. .. 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
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