TP CH SINH HC, 2012, 34(3): 347-353

BIU HIN V TINH SCH PROTEIN TI T HP NLI-IF T T BO Escherichia coli

Nguyn Duy Phng, Najaren Tuteja, L Huy Hm, Phm Xun Hi* Vin Di truyn nng nghip, (*)xuanhoi.pham@gmail.com
TM TT: phc v mc ch nghin cu mc biu hin trong cy chuyn gen ca gen m nhn t phin m NLI-IF (Nuclear LIM Interactor-Interacting Factor) lin quan ti kh nng p ng hn la cng nh nghin cu kh nng bm c hiu vi ADN in vitro ca NLI-IF, chng ti biu hin v tinh sch protein NLI-IF ti t hp trong vi khun E. coli chng DE3. Trnh t m ha NLI-IF c kch thc 1,3 kb trong vector nhn dng pGEMT/NLI-IF c ct ra v a vo vector biu hin pET28a ti v tr EcoRI v XhoI. Vector ti t hp pET28a/NLI-IF c bin np vo t bo Escherichia coli Rossetta. Protein c NLI-IF kch thc 52 kDa c biu hin ti u iu kin nui cy t bo 20oC, nng cht cm ng IPTG 0,1 mM trong thi gian 5h. S dng ct sc k i lc Ni-NTA c kh nng to lin kt gia phc hp Nickel v ui His-Tag ca protein dung hp, chng ti tinh sch c NLI-IF ti t hp t dch chit t bo. Protein tinh sch lin kt c hiu vi khng th anti-His-tag trong th nghim lai thm tch min dch (Western Blot). T kha: Escherichia coli, chu hn, nhn t phin m, NLI, protein ti t hp, vector biu hin. M U

Hng nghin cu v stress thc vt trn th gii hin nay ang tp trung vo vic phn lp v nghin cu c tnh mt tp hp y cc gen lin quan n bt li mi trng v mi lin h gia cc bt li mn, hn v nhit . Hng trm gen c cm ng trong cc iu kin bt li khc nhau v sn phm ca cc gen cm ng vi iu kin bt li c chia lm hai nhm: (1) nhm cc protein chc nng gip thc vt chng li bt li ca mi trng v (2) nhm cc protein iu khin lm nhim v iu ha biu hin gen v truyn tn hiu trong qu trnh p ng iu kin bt li. Cc nhn t phin m thuc nhm th hai v l h gen ln [10]. Gn y, rt nhiu nghin cu v nhn t phin m c thc hin tr n cy m hnh Arabidopsis v cc loi thc vt khc chng minh vai tr quan trng ca chng trong qu trnh iu ha phn ng ca thc vt trong cc iu kin bt li mi trng. Thc nghim chng minh, s biu hin ca cc nhn t phin m kch hot s biu hin ca rt nhiu gen chc nng, iu ny lm tng cng kh nng chu hn thc vt. V vy, cc nghin cu v cc gen m ha nhn t phin m lin quan n tnh chu hn ang tr thnh nh hng nghin cu y tim nng trong vic chn to ging chu hn.

c im c trng ca cc protein iu khin (nhn t phin m) l c hai vng hot ng (domain): (1) vng hot ho cc protein chc nng (activation domain) v (2) vng lin kt (binding domain) vi cc trt t ADN c hiu (cis-acting element) trn vng iu khin ca gen (promoter). Da vo c tnh bm ADN, k thut sng lc php lai n trong t bo nm men c hnh thnh phn lp cc nhn t phin m. Nhn t phin m u tin (OLF-1) c phn lp bng k thut sng lc php lai n trong t bo nm men [15] v ngay lp tc tr thnh phng php y tim nng trong vic phn lp cc gen m ha cc protein c kh nng bm ADN [16]. thc vt, trt t ADN c hiu ABRE (ABA responsive element - yu t p ng ABA) c trnh t li l ACGTGGC ln u tin c pht hin trn vng iu khin gen Em la m [4]. Hai nhm protein iu khin qu trnh phin m AREB/ABF bm vo trt t ADN c hiu ABRE trn cc vng iu khin gen v hot ha s biu hin cc gen chc nng lin quan n khng hn [3, 14]. Tip theo, trt t ADN c hiu DRE/CRT (Dehydration Responsive Element/C repeat - yu t/on C lp li p ng hn) c trnh t li l A/GCCGAC, c pht hin trn vng iu khin gen RD29 Arabidopsis [16] v sau ny 347

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c pht hin trn rt nhiu on iu khin gen ca cc gen chc nng biu hin trong iu kin hn, mn, lnh [2, 5, 12]. Cc gen iu khin qu trnh phin m thuc nhm AP2 (APETALA2)/ethylene-responsive elementbinding factor (ERF) bm vo trt t ADN c hiu DRE v c t tn l DREB1/CBF v DREB2 [6]. Trt t ADN c hiu MYC v MYB c trnh t li l CANNTG (MYC) v C/TAACNA/G (MYB), c pht hin trn vng khi ng gen RD22. Cc nhn t phin m AtMYC v AtMYB bm vo trt t ADN c hiu MYC v MYB v hot ho biu hin gen chc nng lin quan n chu hn [1]. Trt t ADN c hiu nhm gen NAC c trnh t li l CATGTG, c pht hin trn vng khi ng gen ERD1. Ba nhn t phin m h NAC l ANAC019, ANAC055 v ANAC072 bm vo trt t c hiu nhm NAC tr n vng khi ng gen chc nng v hot ha biu hin cc gen ny [13]. Trong mt nghin cu khc, Tran et al (2004) [12]. phn lp c mt gen m ha nhn t phin m lin kt c hiu vi mt trnh t ch tng ng vi trnh t 14 bp nm trong promoter ca gen RPS1, c tn l trnh t ZFHDRS (zinc finger homeodomain recognition sequence). Nhn t phin m ny hot ha mt s gen cm ng vi iu kin stress v lm tng kh nng chng chu stress ca cy chuyn gen [14]. Gn y, s dng k thut sng lc php lai n trong t bo nm men vi trnh t ch c chiu di 50 nucleotid cha trt t DRE trn vng khi ng gen JRC2606, chng ti phn lp c gen m ha nhn t phin m OsRap2.4B t ging la Japonica [8]. Trong mt nghin cu khc, chng ti thit k th vin ADNc t ARN tng s ca ging la Niponpare x l hn v phn lp c gen NLI-IF1 (Nuclear LIM interactor -interacting factor), m ha cho mt protein trung gian nm trong phc hp lin kt protein-protein, thuc h nhn t phin m LIM, tham gia iu ha qu trnh phin m bng phng php sng lc php lai n trong t bo nm men [7]. Kt qu phn tch cy la di (wide type) bng Northern blot v RT-PCR chng t gen NLI-IF tng cng biu hin trong iu kin stress (mn, lnh, mt nc v nhit cao). Cc th nghim lai phn 348

t in vivo trong t bo nm men cng cho thy, protein NLI-IF c kh nng lin kt c hiu vi trnh t ADN ch nm trong trnh t ca 2 promoter JRC0528 v JRC0332 (s liu cha cng b). Trong nghin cu ny, chng ti tin hnh biu hin v tinh sch protein ti t hp NLI-IF trong t bo vi khun E. coli s dng cho nghin cu xc nh kh nng tng tc vi trnh t ADN ich in vitro ca NLI-IF, ng thi phc v mc ch to khng th a dng khng NLI-IF, dng cho nghin cu biu hin ca NLI-IF trong cc th nghim phn tch cy chuyn gen NLI-IF.
PHNG PHP NGHIN CU

Vt liu Trnh t m ha nhn t phin m NLI-IF c tch dng gi trong vector pGEMT. Chng vi khun E. coli Rossetta do Trung tm K thut Di truyn v Cng ngh Sinh hc Quc t (n ) cung cp. Phng php Thit k vector biu hin pET28a/NLI-IF Vector ti t hp pGEMT/NLI-IF v vector biu hin pET28a c x l ng thi vi EcoRI v XhoI. Trnh t m ha NLI-IF v c ghp ni vo vector biu hin nh enzyme T4 Ligase. Biu hin v tinh sch protein NLI-IF Vector ti t hp pET28a/NLI-IF c bin np vo t bo E. coli Rossetta bng phng php sc nhit, chn lc trn mi trng cha kanamycin 50 g/ml, chloramphenicol 34 g/ml. NLI-IF c biu hin vi iu kin nui cy t bo 28oC v 37oC, trong thi gian 3 h v 5 h, c b sung cht cm ng IPTG nng 0,5 mM v 1,0 mM. Protein ti t hp NIL-IF c tinh sch bng ct sc k i lc Ni-NTA (Invitrogen), s dng m y ct cha Imidazol nng 20 mM, 100 mM, 200 mM v 500 mM. Protein tinh sch sau khi thm tch loi mui bng mng cellulose c bo qun -20oC s dng cho cc th nghim tip theo. Thm tch min dch (Western blot) Protein sau khi in di trn gel SDS-PAGE

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c in chuyn ln mng nitroxelluloza theo phng php ca Sambrook et al. (1989) [8] v c nh bng dung dch BSA 1%. Mng nitroxellulose c vi khng th anti-His-tag c gn enzyme AP (Invitrogen) to lin kt protein-protein, sau hin mu bng dung dch c cht p-nitro blue tetrazolium chloride 5-bromo4-chloro-3-indolyl phosphat (NBT/BCIP).
KT QU V THO LUN

Thit k vector biu hin pET28a/NLI-IF Khi phn lp trnh t m ha NLI-IF c kch thc 1320 bp t th vin ADNc, chng ti s dng cp mi c thit k mang 2 v tr nhn bit ca EcoRI v XhoI. Do , a trnh t gen NLI-IF vo vector biu hin pET28a, chng ti x l ng thi 2 vector, pGEMT/NLI-IF v pET28a vi EcoRI v XhoI (hnh 1). Sau khi tinh sch sn phm ct gii hn t gel agarose bng b kit Gel Extraction

Kit (Fermentas), trnh t m ha NIL-IF c chn vo v tr a im ct ca vector biu hin pET28a di tc dng ca enzyme T4 ligase v bin np vo t bo kh bin E. coli chng DH5. Kt qu kim tra khun lc bng PCR (hnh 2A, ging 1-4) cho thy chng ti thu c mt s th bin np mang vector ti t hp pET28a/NLI-IF. Kim tra plasmid tinh sch t cc th bin np ny bng phn ng ct gii hn vi EcoRI v XhoI, chng ti thu c 2 bng ADN c kch thc 1,3 kb (tng ng vi trnh t m ha NIL-IF) v 5,3 kb (tng ng vi khung vector pET28a) (hnh 2B, ging 1-4). PCR kim tra plasmid ny vi cp mi T7Fw/NLI-Rv v cp mi c hiu gen NLIFw/NLI-Rv chng ti thu c sn phm c kch thc tng ng l 1,5 kb (hnh 2C, ging 2) v 1,3 kb (hnh 2C, ging 4) ng vi tnh ton l thuyt. Cc kt qu ny chng t chng ti gn thnh cng trnh t m ha nhn t phin m NLI-IF vo vector biu hin pET28a.

Hnh 1. Kt qu in di sn phm ct gii hn pGEMT/NLI-IF (A) v pET28a (B) bng EcoRI/XhoI

A B C

Hnh 2. Kt qu in di sn phm PCR khun lc (A), ct gii hn plasmid pET28a/NLI-IF bng EcoRI/XhoI (B) v PCR plasmid pET28a/NLI-IF (C).
A. Kt qu in di sn phm PCR t cc khun lc 1-4, vi cp mi c hiu NLI-Fw/NLI-Rv; B. Kt qu in di sn phm ct gii hn plasmid tinh sch t khun lc 1-4 bng EcoRI/XhoI; C. Kt qu in di sn phm PCR vi khun l pET28a/NLI-IF, s dng cp mi T7-Fw/NLI-Rv (ging 1 v 2) v cp mi NLIFw/NLI-Rv (ging 3 v 4).

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Biu hin protein ti t hp NLI-IF trong t bo E. coli Rossetta Chng vi khun E. coli l chng vi khun c thit k mang gen kh tnh c ca promoter T7 s dng trong h thng vector biu hin pET, do chng ti s dng chng vi khun ny biu hin gen NLI-IF c iu khin bi promoter T7. T bo Rossetta sau khi c bin np vector ti t hp pET28a/NLI-IF, c chng ti nui biu hin protein bng mi trng LB lng, cc iu kin nhit 16, 20, 28 v 37oC, c b sung cht cm ng IPTG
A

nng 0,1 mM, 0,5 mM v 1,0 mM, vi thi gian cm ng khc nhau. Kt qu chng ti thu c cho thy iu kin cm ng IPTG 0,1mM, 20oC trong thi gian 5 h l iu kin tt nht biu hin NLI-IF. Qua kt qu in di SDS-PAGE (hnh 3A) cho thy phn on protein thu c iu kin thch hp c kch thc khong 52 kDa, phn on ny khng xut hin mu i chng khng cm ng bng IPTG. Kt qu ny cng ch ra protein ti t hp NLI-IF nm trong c 2 pha, pha cn v pha dch ca dch chit t bo E. coli.

Hnh 3. Kt qu in di SDS-PAGE (A) v Western blot (B) dch chit t bo E. coli Rossetta biu hin gen NLI-IF 20oC, cm ng IPTG nng 0,1 mM, trong thi gian 3 h v 5 h
Kt qu in di pha cn (ging 1-3) v pha dch (ging 4-6) ca dch chit t bo; ging 1, 4: khng cm ng IPTG; ging 2, 5: cm ng IPTG 0,1 mM trong 3 h; ging 3, 6: cm ng IPTG 0,1 mM trong 5 h.

Tinh sch protein ti t hp NLI-IF Do protein NLI-IF c biu hin ra ch yu nm trong pha dch, v vy, sau khi siu m ph mng t bo thu dch chit, chng ti ly tm loi b ton b phn xc t bo v cho pha dch trc tip i qua ct sc k i lcNiNTA. Do NLI-IF c biu hin bng h thng vector biu hin pET28a nn protein to ra s c dung hp vi 1 trnh t gm 6 amino axit histidine. Trnh t His-tag ny s gip protein ti t hp kt bm vo ct sc k, trong khi cc protein khc s b loi b bng dung dch ra cha Imidazol 30 mM. Sau khi ra sch cc protein to lin kt khng c hiu vi ct sc k, protein NLI-IF c y phn on bng dung dch m cha Imidazol 250 mM. Kt qu thu c trn bn in di SDS-PAGE cho thy protein NLI-IF ti t hp c thu li dng tinh sch, c kch thc 52 kDa, tng ng vi kch thc tnh ton l thuyt. 350

xc nh protein, chng ti biu hin v tinh sch c l protein dung hp NLI-IF/Histag, chng ti tin hnh k thut thm tch min dch (western blot), s dng khng th khng trnh t His-tag vi nng pha long 1:25000. Kt qu cho thy, trong dch chit t bo, chng ti thu c bng protein c kch thc khong 52 kDa, tng ng vi kch thc li thuyt ca NLI-IF (hnh 3B), chng t NLI-IF c biu hin thnh cng trong t bo E. coli Rossetta. Kt qu ny cng ch ra vic s dng ct sc k i lc Ni-NTA cho php chng ti tinh sch c protein ti t hp NLIIF (hnh 4B). Nhm khng nh chnh xc hn protein ti t hp tinh sch c l NLI-IF, chng ti x l dung dch protein tinh sch bng ct sc k Ni-NTA vi Thrombin (Promega) loi ui His-tag, sau tip tc cho qua h thng ct sc k Ni-NTA mc ni tip vi Heparin-Sepharose

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(BioVision). Trong mt cng trnh nghin cu trc y, s dng php lai phn t trong t bo nm men (Yeast One Hybrid), chng ti xc nh c NLI-IF l mt nhn t phin m c kh nng lin kt vi trnh t ADN ch [7],v vy s gn vi ct Heparin-Sepharose. Do , khi s dng h thng ct sc k mc ni tip, cc phn t NLI-IF dung hp vn cn ui Histag s b gi li trong ct Ni-NTA, trong khi NLI-IF b loi ui His-tag s gn vi ct Heparin-Sepharose v s c y ra bng m chit cha NaCl 500 mM. Protein tinh sch sau c in di SDS-PAGE v chuyn ln
A

mng vi khng th anti-His-tag. Kt qu cho thy, ch c protein trc khi x l vi thrombin c kh nng lin kt vi khng th anti-His-tag, trong khi protein sau khi x l v tinh sch li qua h thng ct sc k Ni-NTA mc ni tip Heparin-Sepharose khng c kh nng ny (hnh 5). iu ny chng t chng ti thu c protein NLI-IF ti t hp hon ton tinh sch. Vic tinh sch c NLI-IF bng ct sc k Heparin-Sepharose cng cng c thm cho nghin cu trc y ca chng ti v kh nng lin kt vi ADN ca NLI-IF.

Hnh 4. Kt qu kim tra NLI-IF tinh sch bng ct sc k i lc Ni-NTA

A. Kt qu in di SDS-PAGE cc phn on tinh sch NLI-IF; B. Kt qu thm tch min dch (Western blot) vi khng th anti-His-tag (pha long t l 1: 25000). Ging 1: pha cn; ging 2: pha dch cha tinh sch qua ct Ni-NTA; ging 3: dch qua ct; ging 4: phn on ra ct vi imidazol 30 mM; ging 5-10: cc phn on y ct bng imidazol 250 mM. A B

Hnh 5. Kt qu kim tra NLI-IF tinh sch trc v sau khi x l vi Thrombin.
A. Kt qu in di SDS-PAGE NLI-IF tinh sch; B. Kt qu thm tch min dch (Western blot) vi khng th anti His-tag (pha long t l 1:25000). Ging M: thang chun protein; ging 1: NLI-IF tinh sch qua ct Ni-NTA; ging 2: NLI-IF tinh sch x l bng thrombin v tinh sch li qua h thng ct Ni-NTA mc ni tip Heparin-Sepharose.

Protein ti t hp sau khi c tinh sch s c s dng nghin cu kh nng lin kt ADN c hiu ca NLI-IF v to khng th khng NLI-IF dng cho mc ch phn tch biu hin ca NLI-IF trong cy chuyn gen.

KT LUN Trnh t m ha nhn t phin m NLI-IF kch thc 1,3 kb phn lp t ging la Japonica c thit k vo h thng vector

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biu hin protein pET28a. Protein dung hp c biu hin thnh cng trong chng E. coli Rossetta iu kin nui cy 20oC, cht cm ng IPTG 0,1 mM, trong thi gian 5 h. Protein NLI-IF ti t hp c tinh sch bng ct sc k i lc Ni-NTA vi nng imidazol trong m y ct l 250 mM. Sn phm protein sau khi x l vi thrombin loi ui His-tag v tinh sch li bng h thng ct Ni-NTA mc ni tip Heparin-Sepharose c tinh khit cao, m bo cht lng s dng cho cc nghin cu tip theo. Li cm n: Nghin cu c thc hin theo chng trnh hp tc gia Phng Bnh hc Phn t Thc vt (Vin Di truyn nng nghip) v Trung tm Quc t K thut Di truyn v Cng ngh sinh hc (New Delhi, n ).
TI LIU THAM KHO

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EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEIN NLI-IF FROM Escherichia coli

Nguyen Duy Phuong, Najaren Tuteja, Le Huy Ham, Pham Xuan Hoi Agricultural Genetic Institute SUMMARY
In order to investigate the expression level of NLI-IF (Nuclear LIM Interactor-Interacting Factor) transcription factor - encoding gene which involved in drought tolerance in rice and identiflied in vitro DNA binding ability of NLI-IF, we expressed and purified recombinant protein NLI-IF from E. coli Rossetta. 1,3 kb NLI-IF - encoding sequence was cut from pGEMT/NLI-IF and inserted into EcoRI/XhoI site in MCS of expression vector pET28a. pET28a/NLI-IF was transformed into E. coli Rossetta. 52 kDa recombinant protein NLI-IF was expressed optimally in E. coli using 0.1 mM IPTG as an inducer, at 20oC, for 5 h. We were successful in purification of recombinant protein NLI-IF using Ni-NTA affinity chromatography systerm. Purified protein has an ability of binding, specifically, to anti-His-tag antibody in Westerrn blot assay. Keywords: E. coli, Drought tolerance, expression vector, recombinant protein, transcription factor, NLI.

Ngy nhn bi:9-5-2012

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