TAP CHi KHOA HOC VA CONG NGHE Tap 48, so 1, 2010 Tr. 55-62
NGHIEN CLTU BIEU HIEN INSULIN NGU'OI (y E. coli
BUI TH! H U Y S N , LE V A N PHUNG, PHAN VAN CHI
l . D A T V A N D E
Insulin la mgt hormone quan trgng cd vai tro dieu boa dudng huyet do cac te bao beta trong dao Langerhans cua toyen tuy san xuat [9]. Ham lugng dudng (glucose) trong mau la nguon nang lugng thiet yeu cho co the, neu khong duy tri d mirc binh thudng cd the gay ra nhiing can benh nguy hiem [3]. Khi dudng huyet tang cd the gay ra su bai tiet dudng qua nudc tieu, dan den mat/thieu hut glucose, hien togng nay cdn ggi la benh dai thao dudng [7, 5]. Nguyen nhan la do sir thieu hut insulin hoac vi mgt li do nao do co the khdng the sir dung dugc insulin dan den dudng huyet tang cao va gay dai thao dudng [8]. Benh dai thao dudng dugc chia lam hai loai la type I va type II [10]. Dai thao dudng type 1 la loai benh phu thugc insulin, thudng gap d ngudi tre todi (< 35 tool), liia tool hay gap nhat la 10 - 16 tool. Day la mgt dang benh nang trong do cac te bao cua toyen tuy cd nhiem vu tiet insulin bi khiem khuyet nen co the khong cd insulin de sir dung. Neu khong dieu tti bang each tiem insulin, benh nhan se hon me va to vong [8]. Dai thao dudng type II la loai dai thao dudng khong phu thugc insulin, benh thudng gap d ngudi
> 40 tool, ngudi beo phi, ttong do co the van san xuat insulin nhung vi mgt li do nao dd khong the sir dung dugc nd [4].
Ve cau tnic, d ngudi phan to insulin bao gom hai chuoi polypeptide: chudi A gom 21 axit amin va chuoi B gom 30 axit amin [1, 2]. Hai cau noi disulfide (vi tri A7 - B7, va A20 - B19) dong hda tri nhu hai sgi day huge chat hai chuoi vdi nhau, ttong chuoi A con c6 mgt cau noi disulfide giiia goc A6 va Al 1 dam bao phan to kha on dinh trong khdng gian. Dac biet la ca 3 cau noi nay deu bat bien d tat ca cac dang insulin cua dgng vat.
Hien nay, tinh hinh phat ttien benh dai thao dudng d Viet Nam ciing nhu tren the gidi dang c6 xu hudng tang nhanh. Insulin la hormon dau tien dugc tong hgp nhan tao, dugc san xuat bang cdng nghe DNA tai to hgp phuc -vu dieu tri benh nay [14]. Nam 2005, nhu ciu insulin dimg trong tri benh dai thao dudng udc tinh khoang 4 den 5 tin va du kiln nam 2010 la 16 tin [12].
Nhu cau ve insulin cua the gidi vugt qua con so vai tan/nam va vi the nguon cung cap insulin cho tri benh tieu dudng dang ngay cang thieu hut [13]. Trong bai bao nay, chimg toi trinh bay mgt so ket qua nghien cuu bieu hien va xae dinh insulin ngudi to vector pET 32c (+).
2. VAT LIEU VA PHUONG PHAP 2.1. Vat lieu
Nguon gen ma hda cho insulin chuoi A va B da dugc ddng hda trong vector pET14bInsA/B do nhdm cua PGS.TS Le Van Phimg (Dai hgc Y Ha Ngi) cung cap.
Cac hda chit sir dung cho dien di SDS-PAGE to BioRad; Acetonitrile (ACN) to J.T Barker (My); acid formic (FA), trifluroacetate (TFA) mua to Fluka (Thuy ST); dithiothreitol (DTT),
iodoacetamide (lAA), ammonium bicarbonate (NH4HC03), trypsin to Sigma-Aldrich (My) va mgt so hda chat thong dung dimg trong sinh hgc phan to.
2.2. Phirffng phap nghien cii'u
2.2.1. Thiet ke vector bieu Men cho hai chuoi A vd B insulin
Gen ma hda cho chuoi A cua insulin gom 63 cap nucleotide (toang ung vdi 21 amino acid) da dugc ddng hda vao vector pET-14b cd chira diem cat ciia hai enzyme gidi ban Ndel va BamHl d hai dau gen va doan His-Tag (6xHis) d dau N cua chudi A. Gen ma hoa cho chuoi B gom 90 cap nucleotide (toong ung vdi 30 amino acid) cung dugc thilt k l toong tu. Do kich thudc moi gen rat nhd (60 va 90 bp) nen protein dang dung hgp His-Tag chain A va B bieu hien rat khd khan va hau bet ton tai d ttang thai khong tan. De khac phuc nhugc diem nay, cac doan gen lan lugt dugc chuyen vao vector pET-32c(+) bang hai enzyme gidi ban A'col va BamHl tao ra protein dang dung hgp mdi Trx.Tag-His.Tag-Thrombin- S.Tag-Eterokinase-Chain A/B- His.Tag. Theo tinh toan li thuyet dung hgp protein mdri cd kich thudc 22 va 23 kDa. Cau tnic nay lam tang kha nang boa tan nhd noi ghep voi Thioredoxin (Trx) va tang kich thudc phan to, tao dieu kien thuan lgi cho cac qua trinh tinh che tiep theo (hinh 1).
(Chuwn viin pET32c liano Ncol va Baniliri J"iio (long fUile ImAjB).
Phan tich nhan dang bSng khoi pho
Hinh 1. Mo hinh quy trinh thiet ke, bieu hien va xae dinh insulin Quy trinh thi nghiem dugc mo ta torn tat nhu sau:
Plasmid tai to hgp pET14blnsA/B c6 chira gen ma hda cho chuoi A/B cua insulin va vector bieu hien pET 32c(+) dugc xir li lan lugt bang hai enzim gidi ban Ncol va BamHl. Phan img dugc thuc hien d 37°C trong 3 gid. Dien di san phim thu dugc tren gel agarose 1,5% su dimg kit thoi gel QIAGEN dl thu lai bang DNA ma hda cho chuli A, B. Sau do doan gen ma nay nli ghep vdi vector pET 32c(+) da md vdng vdi su cd mat cua enzim noi T4 ligase, phan ung dugc thuc hien d 22°C trong khoang thdi gian 16 gid. San phim phan ung dugc biln nap vao tl bao kha biln DH5a, dugc nuoi ciy, tach chiit plasmid. Kit qua cua qua trinh biln nap dugc kilm tra lai bing phan irng cit bdi enzim gidi ban Ndel, khing dinh doan gen mong muln da dugc dua vao vecto.
2.2.2. Boc trinh tu gien
Trinh tir DNA cua hai doan gien ma hda cho hai chuoi A va B cua insulin dugc tien hanh xae dinh tren may dgc tiinh tu tiJ dgng ABl PRISM® 3100 Avant Genetic Analyzer (Applied Biosystems).
2.2.3. Bieu Men insulin chudi A vd B dudi dgng dung hgp
Vector tai to hgp (sau khi da kilm tra bang phan img cat vdi Ndel) dugc bien nap vao chiing E. coU BL21 (DE3) tao thanh ddng t l bao BL21 (DE3)/pET32cInsA/B vdi dung hgp protein mdi Trx.Tag-His.Tag-Thrombin- S.Tag-Eterokinase-Chain A/B- His.Tag. Dong te bao nay dugc nuoi ciy tiong moi trudng LB long cd bo sung ampicilin (nong do cudi cung la lOOpg/ml), lie 200 vdng/phiit d 37°C. Khi gia tri ODeoo nm cua dich nuoi cay dat 0,5, bo sung IPTG vai ning do culi cung la 1 mM. Dien di SDS-PAGE tren gel polyacrylamid 12,6% de kiem tra ket qua bieu hien.
2.2.4. Xdc dinh chuoi A. B insulin bdng phuang phdp khoi pho
Bang protein insulin tai td hgp (chuoi A va B dung hgp) tren ban dien di SDS-PAGE dugc cit ra va xir li vdi dung dich nia (50 mM NH4HCO3, pH 8,0, 50% ACN). Sau do, cac manh gel dugc khir bing dung dich DTT 5 mM d 56°C, trong 1 gid va alkyl hda bang dung dich lAA 5 mM trong toi, 1 gid d nhiet do phdng. Trypsin (Sigma) dugc them vao vdi ti le enzim : co chat la 1 : 50, d 37°C, qua dem.
Hon hgp peptide sau thuy phan dugc phan tach tren he sac ki long nano (IDnanoLC) vdi cot ngugc pha RP C18 va phan tich tren he may khdi pho QSTAR® XL vdi su ho trg cua phan mem Analyst QS de ghi pho MS va MS/MS. Phan mem Mascot vl.8 dua tren thuat toan Mowse [6] dugc sil' dung de nhan dien protein tren co sd dii lieu NCBInr va Swiss-Prot theo quy trinh da dugc mo ta [11].
3. KET QUA VA THAO LUAN 3.1. Ket qua cat plasmid tai to hop pET32cInsA/Bbang Ndel
Do chuoi A (InsA) va chuoi B (InsB) cua insulin dugc dua vao pET-14b bdi hai enzyme Ndel va BamHl nen qua trinh chuyen doan DNA cd chiia gen A/B vao vector pET-32c se thanh cong neu trong vector tai to hgp mdi cd them 1 diem cat cua Ndel. Trong vector pET-32c da cd san 2 diem cat cua Ndel tai vi tri 396 va 691, doan DNA dugc chen vao to vi tri 198 (Ncol) den vi tri 212 (BamHl) lam tang them 1 diem cat cho Ndel. Theo tinh toan li thuyet thi khi xir li vector tai td hgp mai bdi Ndel se cho ra 3 bang vdi kich thudc toong img la 194 bp, 345 bp va vector gdc 5900 bp.
Ket qua cua phan img cat plasmid tai to hgp bdi Ndel dugc the hien tren hinh 2 cho thay, sau khi xu li bang enzim Ndel, trong so cac plasmid 1 - 8 (InsA trong pET-32c) thi chi c6 ddng 2, 6, 7, 8 va 9 (InsB trong pET-32c) deu cho ra 4 bang cd kich thudc toong duang nhau. Bang tren cung la vector goc pET-32c cd kich thudc khoang 5900 bp, tiep do la hai bang 194 bp va 345bp phii hgp vdi tinh toan. Tren hinh 2 cdn xuat hien them mgt bang 540 bp, cd the day la bang DNA tdng cua 2 bang 194 bp va 345 bp do chua dugc cat hoan toan. Nhu vay doan DNA mong muln da dugc chen vao vector dich. Phan tich ket qua xae dinh trinh tu nucleotide gen ma
hda cho chuli A va B cua insulin tren may dgc tiinh tu tu dgng ABI PRISM® 3100 Avant Genetic Analyzer cho thay cd su toong dong 100% vdi trinh tir chuin chung td vector da dugc thilt k l thanh cong (ket qua khong trinh bay d day).
1-8: liu.\l-S Ul pET .i: c U MiUktfi I Kb " Ii«Bl ui pET .^: c
Hinh 2. Kit qua cat plasmid tai to hgp pET32cInsA/B bang Ndel
3.2. Biiu hien chu6i A va B cua insulin dang dung hop d E. coli
Kit qua bilu hien chuoi A va B cua insulin dudi dang dung hgp dugc kiem tra bang dien di SDS-PAGE tren hinh 3a cho thay, trong dich chiet tong so cua cac ddng te bao thu lai d thdi diem 3h sau cam iing (A3, B3) cd xuat hien mgt bang protein mdi so vdi mau dich chiet te bao khdng dugc cam ung bdi IPTG (AO va BO). So vdi thang M chuan, sau 3 gid cam img P3 xuat hien 1 bang protein c6 kich thudc khoang 23 kDa, A3 cung xuat hien mgt bang cd kich thudc khoang 22,0 kDa (do doan DNA 120 bp cd chira gen ma hda cho chuoi A chen vao) va B3 xuat hien bang protein mdi khoang 23 kDa. Ket qua nay phu hgp vdi tinh toan li thuyet. Nhu vay he bieu hien pET 32c mang to hgp gen InsA (InsB) da boat dgng on dinh de bieu hien protein lai mdi.
a) PO P3 AO A3 A3 B3 B3
b)
116 '
2 kDa kDa
18.4 14-4 ^ ,
Hinh 3. Dien di kilm tra bilu hien dung hgp protein chuoi A/B tren gel polyacrylamide 12,6%
a) Bilu hien cac dong te bao mang gen A/B; b) Kiem tra do tan cua cac dong te bao da bilu hien M: Marker SM 0431 (Fennentas); PO: dong te bao chira vector nguyen the truoc khi cam iing;
AO, BO: cac dong tl bao chiia gien A/B trudc khi cam img; P3, A3 va B3: cac dong te bao sau khi cam iing;
At, Bt: dich tl bao ting sl; As, Bs: dich tan; Ai, Bi: phan khong tan
Chung toi tilp toc khao sat tinh tan cua protein mdi (hinh 3b). Mac du bieu hien protein dudi dang thi vui ciing cd mgt s l uu dilm nhit dinh nhung protein tao ra thudng bi niat boat tinh sinh hgc va qua tiinh tai tao lai ciu tnic toong dli phiic tap va t i n kem. Hon niia de co the tinh sach dugc protein de dang thi cin phai bilu hien protein dudi dang hda tan. Trong thi nghiem nay cac dong tl bao dugc cam ung a 22°C khoang 6 gid khi ODeoo cua dich t l bao dat 0,6 -0,8. Kit qua thi hien tren hinh 3b cho thiy protein d dang tan (As va Bs) chilm ty le rat ldn va lugng protein ton tai d trang thai khdng tan (Ai va Bi) la khong dang k l so vdi protein ting so (At va Bt).
3.3. Nhan dien insulin tai to hop
Bing phuang phap IDnanoLC-ESl-MS/MS, p h i khii cua cac peptide dugc tien hanh nhan dien tren ca sd NCBInr vdi su h i trg cua phan mem Mascot vl.8. Diem dang chu y d day la chuli A vdi 21 amino acid khong cd diem cat (sau K va R) cua trypsin nen khdng nhan dien dugc cac manh peptide d chuli A ma chi nhan dien dugc cac manh peptide cua thioredoxin- Trx ding bilu hien (kit qua khong trinh bay d day). Trong khi do, chuoi B cd hai diem cat cua trypsin nen ket qua nhan dien dugc minh hga nhu hinh 4.
{^CUNM} Mascot Search Results
User E.mail Search title MS data file Database Taxonomy Tdjitesta-mp Significant hits
Tran The Thanh tranthethanhgibt.ac.vn
d:\EE Sciex data\Projects\training\Data\Services\7 Feb 07SETl.wiff ; Bl C: \WIMNT\Prof iles\aDMINI~l\L0CMiS~l\Te.'np\.'rias2B. tanp
NCBInr 20050514 (2336311 sequences; 791794155 residues) Homo sapiens (huiian) (129146 sequences)
6 Apr 2007 at 11:46:54 GMT
g i 142543181 Chain B, l g e Fv Spe7 Coraplexed With a Recoinbinant Thioredoxin a i l 1127137 Chain B, Nmr S t r u c t u r e Of The Hu.man Insulin-His(B16)
Hinh 4. Ket qua nhan dien insulin chuoi B dang dung hgp vai thioredoxin
Ket qua tten cho thay, khi phan tich nhan dang bang khoi pho trong bang bieu hien (khoang 23 kDa) cd hai protein dugc phat hien la thioredoxin va chuoi B cua insulin cd gan duoi His, ket qua nay phu hgp vdi thiet ke vector li thuyet. Diem so (score) cua thioredoxin va chudi B insulin lan lugt la 177 va 65 (hinh 5).
Probability Based Alowse Score
Score is - 1 0 * L o g ( P ) , w h e r e P is tiie piobabilitv- tliat the o b s e n e d matcli is a laiidom e\eiit.
Individual ions s c o r e s '^55 indicate identit>' or e x t e n s i \ e homology ( p < 0 . 0 5 ) .
; 3 5 -
1 5 T
1 0 - Chu6i B insulin
FVNQHXiCGSH L V E A L H L V C O E R G F E ^ Y X P K T F F S T T P K G F F X X P K T >
Thioredoxin
100 ISC P r o b a b i 1 ttM Based rowse Score
Hinh 5. D i e m so nhan dien protein theo thuat toan M o w s e
Trinh tu amino acid chuoi B insulin cho th4y chi co hai dilm cit (tai vi tri B22 va B29) khi thuy phan b5ng trypsin, tao thanh hai manh peptide la GFFYTPK va GFFYTPKT. Khi phan tich khii phi trinh tu cua hai manh nay deu dugc phat hien (mau dd dam).
+TOF MS: Experiment 1, 60.273 minIrom Sample 1|El|ol7 Feb 07£ET1.™f(
at35B7121775301B2170e-004.lO=4e05l74S10B896S230e+001 Ion m/z = 430 05; z ^ +2
578 05GD
opzs '
51 1.1184
200 300 400
, ..502 722B
500 600 700 800 900 1000 1100 1200
7 1104
y7 y6 V5y4y3y2 yi
7 0257 \ 2
>2^244 0;53
° £ U 6 7 1 ^
G55 1505
^ y 6 ^ 802,4132
200 300 400 500 600 700 900 1000 1100 1200
m/z. amu
Monoisotopic mass of neutral peptide (Mr): 858.43 Fixed modifications: Carbamidomethyl (C)
Ions Score: 34 Matches (Bold Red): 5/48 B
# a a""" b b"^ Seq. y y++ y* y*'"' § 1: 30.03 l\P. 58.03 29.?2 G : { 2 177.10 89.06 205.10103.05; F 802.41:401.7l|785.39 393.20 6 J 324.17Jl62.59ij52.17176,59| F 655.55 328.18|6k32^9.66i5|
4 487.23 2-H.12 515.23258.12 Y 508.28 254.64 491.25 246.13 4 5 588.28 294.65 616.28 308.64 T 345.21173.11328.19164.60}
6685,33 343,r713.33357.17 p 244.1' 122,59227.14114.072
' l l l l
K il47.1lj 74.061130,09! 65,55 1Hinh 6. A. Pho khoi cua ion m/z = 430,05 amu; B. Giai trinh tu peptide GFFYTPK
Phan tich pho khoi trinh tu manh ion peptide GFFYTPK, chiing toi nhan thay manh nay c6 gia tri m/z = 430,05 amu, z == +2 (m = 858,43 Da). Qua trinh phan manh va giai trinh tu ion peptide GFFYTPK dugc minh hoa nhu hinh 6.
3.4. Xae dinh khoi lirong cua insulin chuoi A va B
Mw= 24007 Da 103d 100O eoo
800 700-
0 0 0 •
2 0 O -
b)
, + 4
Mw 3561 0 a
. — ^ « ' ^ - ^ ""'^'tii, uy,.
,J^
Hinh 7. Xae dinh khii lugng phan tu cua insuhn chuoi A (a) va chuoi B (b) bSng phuang phap ESI-Q-TOF
Chu6i A va B cua insulin duai dang dung hgp dugc xii li vai enterokinase nham loai bo thioredoxin va duoi histidine. Sau khi xu li, hai chuoi A va B cua insulin dugc xae dinh khoi lugng bang he th6ng kh6i ph6 ESI-Q-TOF (Electrospray-Quadruple-Time of Flight) (Hinh 4).
Ph6 TOF-MS cho thky chuoi A va B ihn lugt co kich thuae la 2400.7 Da va 3561 Da, ket qua nay phu hgp vai li thuy6t do chudi A eo 21 aminoaeid va chuoi B co 31 amino acid va trong te bao E. cod nhom SH cua cystein a dang tu d.o. Nhu vay, vai phuang phap nay chiing toi da khing dinh duge sir bilu hien ciia insulin tai t6 hgp (chuoi A, B), va xae dinh dugc khoi lugng cua hai chuoi rieng biet.
4. KET LUAN
Chu6i A va B cua insulin tai to hgp da dugc bieu hien trong te bao E. coli ehiing BL21(DE3). Cac ket qua xae dinh bSng kh6i pho da khang dinh chuoi B va B ciia insulin tai to hgp da duge bieu hien phu hgp vai tinh toan li thuyet va thiet ke ban dau.
Ldi cam an. Cong trinh dygc thu'c hien tai Phong Thf nghiem Trong diem Cong nghe Gen, Vien Cong nghe Sinh hoc, Vien Khoa hpc va Cong nghe Viet Nam.
TAI LIEU THAM KHAO
1. Bentley G. E., Dodson G. D., Hodgkin D., and Mercola D. - Structure of insulin in 4-zinc insulin, Nature 261(5556) (1976) 166-168.
2. Dunn M. F. - Zinc-ligand interactions modulate assembly and stability of the insulin hexamer - a review, BioMetals 18 (4) (2005) 295-303.
3. Farmer A., Wade A., Goyder E. - Impact of self monitoring of blood glucose in the management of patients with non-insulin treated diabetes: open parallel group randomised trial, BMJ 335 (2007) 132.
4. Harris R., Donahue K., Rathore S. S., Frame P., Woolf S. H., Lohr K. N. - Screening adults for type 2 diabetes: a review of the evidence for the U.S. Preventive Services Task Force, Ann. Intern. Med. 138 (3) (2003) 215-29.
5. Louise G. J. - Diabetes, Atlanta: Centres for Disease Control, 2007.
6. Pappin D. J., Hojrup P., Bleasby A. J. - Rapid identification of proteins by peptide-mass fingerprinting, Curr. Biol. 3 (6) (1993) 327-332.
7. Pugliese G., Zanuso S., Alessi E., Simonelli P., Fallueca S., Fallucca F., Balducci S. - Self glucose monitoring and physical exercise in diabetes. Diabetes Metab. Res. Rev. 25 (2009)11-17.
8. Rother K. I. - Diabetes Treatment — Bridging the Divide, N. Engl J. Med. 356 (15) (2007) 1499-1501.
9. Steiner D. F. and James D. E. - Cellular and molecular biology of the beta cell, Diabetologia 35 (2) (1992) S41-S48.
10. Tiemey L. M., McPhee S. J., Papadakis M. A. - Current medical Diagnosis & Treatment, Intemahonal edition. New York: Lange Medical Books/McGraw-Hill. pp. 1203-15. ISBN 0-07-137688-7,2002.
11. Tran T. T., Nguyen T. M. P., Nguyen B. N., Phan V. C. - Changes of serum glycoproteins in lung cancer patients, Joumal of Proteomics and Bioinformatics JPB 1(1) (2008) 15-21.
12. www.annals.org/cgi/pmidlookup?view=long&pmid=12558362. Retrieved on 2008-07-19.
13. www.cdc.gov/nccdphp/publications/aag/ddt.htm. Retrieved on 2007-09-14
14. Xu B., S. Q. Hu, Y. C. Chu, S. Wang, R. Y. Wang, S. H. Nakagawa, P. G. Katsoyannis and M. Weiss - Diabetes-associated mutations in insulin idenhfy invariant receptor contacts, Diabetes 53 (6) (2004) 1599-602.
SUMMARY
STUDYING ON EXPRESSION AND IDENTIFICATION OF HUMAN INSULIN IN E. coU Insulin is a hormone that produced by beta cells in the pancreas and regulated the glucose blood levels. Briefly, our strategy for the gene construction and expresion is described as follow.
pET14bInsA vector contained gene A that encoding for the A peptide chain and pET14bInsB vector contained gene B that encoding for the B peptide chain. These vectors and the expression pET-32c(+) vector were digested by Ncol and BamHl. The released insulin A/B fragment and the opened plasmid pET-32c(+) were purified and ligated by T4 DNA ligase at 22°C, overnight.
The resulting recombinant plasmid pET32cInsA/B was transformed into the E. coli BL21(DE3) strain. The expression of the fusion protein was observed by induction with 1 mM IPTG and analysed by SDS-PAGE. Two new protein bands with the size of aproximately 22 kDa (equvalent the size equvalent the size of ftision A) and 23 kDa (the size of fiision B) were then excised and digested with trypsin overnight. The results of identifying by NanoLC-MS/MS (Nano Liquid Chromatography - Tandem Mass Spectrometry) showed that the recombinant protein was truely the fusion A(B)-Trx that contained different peptides from chain A/B and thioredoxin. This result is an evidence to confirm the suecessflil construction and expression of chain A/B of insulin in the fiision form in E. coli with pET-32c(+) vector.
Key words. Insulin; pET-32 e(+); E. coU BL21 (DE3); NanoLC-MS/MS
Bia chi:
Bui Thi Huyen, Phan Van Chi
Vien Cong nghe sinh hgc, Vien KH&CN VN.
Le Van Phiing, Dai hoc Y Ha Noi.
Nhdn bdi ngdy 12 thdng 8 ndm 2008
