Science & Technology Development, Vol 17, Ne.T4-2014 Tao clang, bi4u hiOn va tinh sach protein hGM-CSF (Human Granulocyte - Macrophage Colony - Stimulating Factor) chu chat to bao Escherichia coli • Nguyen Phu Sang • Nguyen Thanh Thao • Tran Van Hieu Tru,o,ng Dal hoc Khoa hoc Ty' nhien, DHQG - HCM ( Ben nkin 30 thong 10 ndm 2014, nhdn dang 16 (hang 01 nam 2015) TOM TAT hGM-CSF /à met cytokine bao gem 127 amino acid, vol vj tri cystein hinh cau n6i disulfide Mac de, dang tw nhien cOa hGM-CSF la glycoprotein nhung stir glycosyl h6a kh6ng c6 anh huerng den chirc nang sinh h9c caa protein GM-CSF c6 cherc nang kith thich sw ten tai, tang sinh, biet h6a cOa cac to bao tien than No mau va tang ctrang chuv nang c0a cac to bac, mau twang GM-CSF (15 dut;rc sur dyng rang rai de diet, trj nhieu loci benh nhu': chYng suy giam bpch cau trung tinh hea tri ung thy se, dyng coy ghep fay xtrang, benh suy giam bpch ceu tOy cep Trong nghien cOv thong tai trinh bay kat qua too dang va bleu hien protein hGM-CSF chu chat to bac) Escherichia coli Gen ma h6a cho GM-CSF ngtrai 'Ivey nhan ban bang phtrang phap PCR voi cap mei CO mang trinh tu' cat giol hpn cOa Ncol va Xhol Dopn DNA khuech dpi cluvc dang h6a tong vat) vi tri Ncol/Xhol fret) vector pET- 22b va dyer chen clOng khung vai trinh tin MO tiet pelB Vector bleu hien pET-hGM duer bien nap vac) to bao E coli 8L21(DE3) va the bien nap dtrac cam Ong IPTG de kiem tra kha nang bleu hien protein hGMCSF Phan dopn protein chu chef duix thu nhan bang phuang phap sec them than Sir bleu hien cOa hGM-CSF duet kiem tra bang dien di SDS-PAGE cac phan Clopn protein king, to bac) chef va chu chef Protein phan dopn chu chat sau (96 &rot tinh the bang sec ky trao del anion sir dyng cat Q Sepharose FF voi phyang phap dung ly tang nang mu6i Ket qua dien di SDS-PAGE cho they c6 sw hien dien vpch protein twang dvang vai hGM-CSF d pha chu chef cua thong E coli BL21(DE3)/pET-hGM va protein thu nhan claw qua boat sec V dat de tinh sach 97,4 % Ket qua bieu hien va tinh sach tren da dyer khang djnh bang phvang phap Western Blot II& khang the dac hieu khang hGM-CSF Ter kh6a: GM-CSF, chu chef, sec ky trao d6i anion Trang 12 TAP CHI PHAT TRItti KRIM TAP 17, SO T4-2014 Mlle DAU Nhan to kich thich tao khem bach cau hat-dai thuc bao nguai — hGM-CSF (human Colony-Stimulating Granulocyte-Macrophage Factor) — la met cytokin ce ban chat la glycoprotein, gem 127 amino acid c6 tong lueng phan tir tir 14 den 35 kDa tit), theo tinh tang dui:mg hea GM-CSF oft hai vj tri N-glycosyl hea va ben vj tri 0-glycosyl hea, nhien su glycosyl hea khong anh twang den hoot tinh sinh hoc cia protein [1] GM-CSF CO hai cau nei disulfide net phan to hinh gitta cite gec a not Cys54 va Cys96, Cys88 va Cys121 Cu disulfide the nhat (54-96) lien quan den hoot tinh sinh hoc cia GM-CSF cau not disulfide lai khong co firth hue:mg tort hoat tinh [2] GM-CSF nguiri tai to hqp da duqc sir dung rang Ili de dieu tri cac benh nhu suy giam bach cau trung tinh, benh bach eau toy cap, sir dung city ghep to bao g6c tao mau hay lam to duqc vac-xin Cho den nay, cytokin can duqc chi dinh cho clic benh nhan bj suy giam mien djch, AIDS hay bj nhiem Ming [3, 4] Nhu vay, vii tiem nang ling dung ceng them nhu cau sir dung met nhieu, hGM-CSF la met nlang protein tit to hqp dang duqc quan tam nghien ceu va san xuat hien nay.Trong cac he thong duqc sir dung a bleu hien protein tai to hqp thi E toll IA met he thong duqc sir dung kha phe bieu Protein tai to hqp ce the duqc bieu hien cac dang khac va cac khong gian khac a to bao E colt t3 day thong toi lua chon tiet vio chu chit vi no co nheng uu diem phO hqp vei protein muc tieu nhu san pham tao thuerng a dang tan va khong chira methionine dau N, vOng chu chat tao mot truang thuan Iqi cho viec hinh cau not disulfide [5, 6] Trong Ink bio nay, chimg toi trinh bay cac ket qui deng hea va bieu hien hGM-CSF chu chat to bio E colt nhtr la buOc nen tang ban din quy trinh nghien cOu nham san xuAt protein VAT LBW VA PHUVING PliAP Chiang vi sinh vat va plasmid endA Chitng vi khuan E colt DH5a hsdR17 (rk-/mk) supE44 thi X recAl gyrA96 AlacU169 (080 lacZ cIM15)] (Takara) duqc sir dung de tao deng va km trft plasmid E colt ompT, hsdS (rr mB), gal (DE3)] BL21(DE3) duqc sir dung lam chOng chit de bieu hien protein muc tieu Plasmid pET-22b (Novagen), kich thtrec 5493 by china promoter T7 duqc cam Ong bang IPTG(isopropyl I3-D thiogalactoside), gen khang ampicillin (Amp) duqc ding de tao plasmid pEThGM bieu hien hGM-CSF Plasmid pET-22b co met trinh to tiet pelB dau N, gen muc tieu &cm then deng khung yeti trinh to nay, protein duqc tao thimh se duqc tiet vio vimg chu chat to No E colt Tich chit plasmid Cac plasmid duqc tech chi& theo phtrong phap SDS-kiem theo mo to cita Sambrook va Russell [7] Thiet kg plasmid tai to Ito plET-hGM mang gen hgm-csf ml boa cho protein hGM-CSF Gen hgm-csf ma hea cho protein hGM-CSF duqc thu nhan tir plasmid pGM-CSF (cung cap ben BM Cong nghe Sinh hoc Phan tO - Mai outing, Tnrang Doi hoc Khoa hoc To nhien TPHCM) bang each PCR vOi hai mit 5'-Nco va 3'-Xho theo chtrang trinh 95 °C15 phut; 30 chu ky: 94 °C/45 phid, 55 °C/45 giay, 72 °C/45 phitt; ket thec, 72 °C/10 phut Hai mei se bat cap vao khuon va nhan ban doan trinh to hgm-csf San phiun tao la doan gen co kich thudrc khoing 415 by cling vont vi tri cat gioi hart cOa enzyme Xhol va Ncol hai dau San pham PCR duqc xti ly tao dau dinh vei hai enzyme cat gioi hen tren va tinh the bang EZ-10 Spin Column DNA Gel Extraction kit (BioBasic, Canada) theo huang din cOa nha san xuAt Trang 13 Science & Technology Development, Vol 17, No.T 4-2014 Plasmid pET-22b cling duck; cat ma yang bang cap enzyme Xhol vi Ncol, bet hoat enzyme bang phenol/chloroform va tinh che bing EZ-10 Spin Column PCR Products Purification kit (BioBasic, Canada) Kich thuot cua doan DNA mang gen hgm-csf va plasmid pET-22b da cat mer yang dirge xac dinh bing dien di tren gel agarose % Plasmid pET-hGM dime eau tree bang each A not gen hgm-csf da xu ly veri Xhol va Ncol vao plasmid pET-22b cling dtrac xu ly yeti hai enzyme Tao to bio nap va Min nap plasmid pEThGM vio to bao E coliDH5a Tao to bao E coli kha nap bang phuang phap calcium lanh vi pET-hGM dirge bien nap vao E coil DH5a bang phuang phi!) sec nhiet theo mo to caa Sambrook va Russell [7] Huyen phis to bao sau bien nap dime trii tren moi truing LB agar (tryptone 1%; yeast extract 0,5 %; NaCI 0,5 %; agar %) be sung Amp dat ming da cuOi 100 pg/mL (LBA-Amp100), IA 37 °C, 14 - 16 girl Sing loe the bien nap vi kiem tra bing enzyme eat giai han vi PCR plasmid Cic the bien nap DH5a mac dtrqc tren m8i twang LBA-Amp tiep tuc cluac sang be so be bing phuang phi!) PCR khan lac vii cap mei 5'-Nco va 3'-)Cho Thu nhen the bien nap cho ket qua PCR throng tinh va tach chi& plasmid Sau nen Wirth kiem tra plasmid bing phuang phap PCR vii cap mcii bit cap tren plasmid T7pro/T7ter (Novagen) vi cat giai han vii hai enzyme Xhol va Ncol Plasmid tai to hap pET-hGM thu nhen sau cite qua trinh kiem tra dime tien Minh giai tint' to theo phuang phap Sanger cai tien bed ding ty Macrogene (Han Quec) Ket qua giai trinh At duqc phan tich vi so sink biing phan mem Jellyfish (Lab Valocity) Trang 14 Bleu hi'fn protein hGM-CSF Plasmid pET-hGM dime bien nap vao to bao E coli BL21(DE3) kha nap Chung E coil tii to hap duac cam Ong bieu hien hGM-CSF bing nuoi cay lic m8i twang LB, be sung ampicillin 100 tg/mL a 37 °C, ttic de lic 250 rpm Khi ODA dat 0,8; be sung IPTG den neng da cuii IA 0,5 mM; tiep tuc nu& lee 6.30 °C 16 h Tach cac phan doan protein bing phuang phap sec them that' theo m8 ti dui Ausubel vi ceng su [8] Phin tich SDS-PAGE va lai Western Cac phan doan protein king, to bao chat, chu chat thu &roc se &me dien di tren gel polyacrylamide 12,5 % co su hien dien cua SDS vi so sanh kich thuac vii thang protein chuen Protein sau dien di dtrac chuyen len ming lai vi thirc hien lai Western blot yeti khan the chuet don clang khang hGM-CSF vi phat hien nher khang the khang IgG cua chuert ceng hop veri horseradish peroxidase HRP Hien phim bang be Kit ECL (Amersham) Tinh sack protein hGM-CSF hing sic ki trao doi anion Do hGM-CSF co pl = 5,5 a pH = 7,5 protein tii to hap hGM-CSF tich dien am nen phucrng phap sic kjr trao dcii anion dime sir dung de tinh che Dung dich can bing (ddA): NaCI 20 mM-tris 50 mM, pH 7,5 Dung dich dung ly (ddB): Na2HPO4/NaR2PO4 0,1 M, NaCI M, pH 7,5 He thong tinh che AKTA FPLC dugc can bang bing mac Cat tinh che anion Q SP FF ml (GE Healthcare) dtrac rira bang mac, can Wing ben dung dich A Mk dim nap vio cat yeti Mc de (long mUphat, tai can bing cat bang dung dich A Dung ly theo bee thang veri tic de clang mL/phtit, 20 % dung dich B va 80 % dung dich A ttrang to vii 100 % B Thu nhen plan doan protein tuong ling vii 20 % va 100 % dung dich B Phan tich ket qua bing dien di SDS-PAGE va lai \red kiting the dee hieu khang hGM-CSF TAP CHI PHAT TRIO' KWH, TAP 17, S0 T4-2014 KET QUA VA BIN LUAN ly too deu dinh bang cap enzyme Xhol va Ncol San pham khuech dai gen hgm-csf/ XhoI va Ncol (km tinh the va net vac, plasmid pET-22b da cat mo yang vai ding cap enzyme XhoI va (Hinh 1B) HO'n hop san pham not duce bien nap vao E coli DH5a Ira sang loc bang mOi truing LBA-Amp Tao vector tii to hqp pET-hGM mang gen hgm-csf Gen hgm-csf &roc thu nhan bang phuong phi!, PCR tir plasmid pGM-CSF cho san pham co kich thtroc 415bp Mang fmg vai kich Muck du kien (Hinh 1A) San pliant PCR sau &roc xtr WI hp 41$ bp ,4 Binh Thu dan gen hgm-csf va plasmid pET22b A, Ket qua thu nhan gen hgm-csf dY plasmid pGM-CSF 1, Thang chuim DNA 1kb plus; 2, San pham PCR thu nhim gen hgm-csf hY plasmid pGM-CSF.B, Ket qua kiem tra plasmid pET-22b 1, Thang chuan DNA lkb plus; 2, Plasmid thu titian duce cat bang enzyme XhoI va NcoI Ifinla Ket qua PCR khuan lac du tuyen 1, Thang chuen DNA 1kb plus;2, San pham PCR khuan lac E coli DH5a (dad chimg am); 3, Sim pham PCR vdi khu8n DNA la plasmid pGM-CSF (di chfmg duong); 4, Sim pham PCRkhutin lac du tuyen The bien nap moc duck ten mOi trueing dtmc sang Inc so 6'0 bang phuong phip PCR khan lac vOi cap mOi 5'-Nco/3'-Xho Ket qua dien di kiem tra ten gel agarose 1% tren Hinh cho they san pham IChuach dai la mit each co kich thuerc 415 by Mang Ong vai kich thtrac gen hgm-csf Khuim lac duct tech plasmid, tiep tic kiem tra plasmid tai to hop bang phuong phip PCR plasmid va cat giOi hon O Hinh 3A, gieng 5, san pham PCR plasmid pET-hGM v6i cap mOi 5'-Nco/3'-Xho cho they xuet hien Wit vach co kich think khoang 415 bp, Mang Ong vdri kich think gen hgm-csf Nhu vay, plasmid tai to hop dang hem duce co su hien dien dia gen hgmcsf Phan Ong PCR veri cap mOi bit cap tren plasmid T7pro/T7ter cho san pham co kich think 678 by (Hinh 3A, gieng 5), bang kich think dorm DNA mang gen hgm-csf gifta hai mei Plasmid tai to hop sau &roc kiem tra bang phuong phip PCR, duce tiep tic kiem tra bang phuong phip cat gicii han Hinh 3B gieng va gieng twang fmg vat plasmid pET-22b Ichong mang gen Ara plasmid tai to hop pET-hGM duos cat mer yang bang Xbal Hinh anh tren bin dien di cho they co su chenh tech kich thuoc gift hai plasmid Ben canh dO, plasmid duce xir ly veri cap enzyme NcoI va XhoI cho hai vach Mang fmg vOi kich think plasmid pET22b (5493bp) va gen hgm-csf (415bp) not vao (Hinh 3B, gieng 4) Nhu vay, co the gen hgm-csf duqc then vao plasmid pET-22b Trang 15 Science & Technology Development, Vol 17, flo.T 4-2014 415 by bio Jri Binh Ket qua kiem tra plasmid bang PCR va cat gieri han A, Ket qua kiem tra plasmid bang PCR 1, Thang chuan DNA 1kb plus;2, San pham PCR plasmid pET-22b v6i cap mei 5'-Nco/3'-Xho; 3, San pham PCR plasmid pET-22b v6i cap mei T7-pro/T7-ter, 4, San pham PCR plasmid tai to hqp pET-hGM v6i cap moi 5'-Nco/3'-Xho; 5, San pham PCR plasmid tai to hop pET-hGM vOi cap moi T7-pro/T7-ter.B, Ket qua kiem tra plasmid tai to hop bang enzyme cat giai han 1, Thang chuan DNA lkb plus; 2, Plasmid pET-22b cat bang XbaI; 3, Plasmid pET-hGM cat bang Xbal; 4, Plasmid pET-hGM cat bang XhoI va Ncol Plasmid sau duoc kiem tra bang phuong phi') PCR va cat gioi han duoc xac dinh c18 chinh /tic ye met trinh to bang each giai trinh to va so sinh vai trinh to ly thuyet bang phan mem Jellyfish Ket qua cho they có so ding khung dich ma vai trinh to tin hieu pelB va tuong &mg 100 % so vol trinh to gen da thiet ke dtra tren ngan hang gen (ket qua khung trinh bay) Qua cac ket qua phan tich tren cho they chimg toi da chon ducic (long vi khan E coli DH5a mang plasmid tai to hop mong muen pEThGM, plasmid duoc bien nap yao E coil BL21(DE3) de too chimg E coil BL2I(DE3)/ pET-hGM bieu hien GM-CSF ngutri tai to hop Bien hien protein hGM-CSF chu chAt to bao E coli BL21(DE3)/pET-hGM De cam Ong so bieu hien dm gen hgm-csf to bao E coli BL21(DE3), chimg toi tien hanh nu8i cay (long to bao moi truOng LB long co b6 sung ampicillin neng dO mit 100 ugh/IL WI cam img IPTG Yeti nOng di5 0,5 mM Sau cam img tir 16-18 h o nhiet cti) 30 °C, Wirth thu sinh to bio Thoc chimg toi hien tech cac phan doan protein bang phtrang [Map sac them than theo m8 ti caa Ausubel va gong so Sau sir dung phuong phap SDSPAGE de phan tich protein thu duoc cac phan clop So hen hien hGM-CSF E coli BL21(DE3)/pET-hGM A, SDS-PAGE; B, Western Blot v6i khang the dac hieu khang hGM-CSF;1, Thang phan to luting thap; 2, 3, 4: E coli BL21(DE3)/pET-hGM cam img bang IPTG (phan doan protein tong, to bit° chat, chu chat); 5, 6, E coli BL21(DE3)/pET-hGM (phan doan protein tOng, to bao chat, chu chat); 8, 9, 10 E coli BL21(DE3) cam img bang IPTG (phan doan protein tOng, to bao chat, chu chat) Trang 16 TAP CHI PHAT TAIEM DM TAP 17, S614-2014 Tren I-linh 4A, clic gieng 5, 6, 7, 8, 9, 10 la cac nghiem thac dai chang De kiem tra stn bieu hien dm protein hGM-CSF sau cam img, so sanh cac vach dm protein yen cac nghieifi thirc &Si chimg Tren bin dien di, cac giLg 2, 3, la mau cac phan doan protein Wong img vOi phfin doan protein tang so, to bio chat, chu chit clia chnng E coil BL21(DE3)/pET-hGM c6 cam *rig IPTG, chang tai !than thiy mL phan doan chu chit c8 sir xuat hien mat vach protein nim gifta vach 14,4 kDa va 20,1 kDa cult thang phan tit luqng, twang Crag vai kich thuac cna protein hGM-CSF ly thuyet (gieng 4) Trong mL protein cac phan doan chu chat a nghiem thirc dai chimg (gieng 7, 10) khang nhan thay sv xuat hien cua vach protein Nhu stay, chimg tai dir doan, da bieu hien dirge protein hGM-CSF chu chit to bao E coil BL21(DE3)/pEThGM De Idling djnh ket qua chi:mg tai bap tut thvc hien phucmg phip Western Blot vai '7 66 yaw khang the (lac hieu khang hGM-CSF Tren ban phim xuat hien vach tin hieu tuong ang yen vi tri cna each protein xuit hien phan doan chu chAt duac cam frog IPTG (Hinh 4B, giLg 4), ket qui khAng dinh vach protein dam xuat hien duo: cam ang chink la GM-CSF Tir nhfIng ket qua ter'chung toi ket Juan da bieu hien ding protein hGM-CSF chu chit to bao E coil BL21(DE3)/pET-hGM Tinh such hGM-CSF chu chit to bio E coil BL21(DE3)/pET-hGM bing sic kI trao di anion Do protein hGM-CSF co pI lchoang 5,5 la mat protein china &lieu nham mang dien tich, tich dien am dieu kien pH sinh ly (6-8) Vi the chi:mg toi thvc hien khao sat tinh sach protein bang sic kY trao dai anion, dung ly bing each thay doi ming de) muSi hay pH vi cutii carig danh gii mire da tinh sach 7 ale 45 30 tale 20.1 14.4 le' Ill • Hlnh Kat qua tinh sach hGM-CSF bang phueng phap sac ky trao dai anion (cAt Q Sepharose FF), dung ly bang tang nang d4 muai A, SDS-PAGE; B, Western Blot vai khang the dac hieu khang hGM-CSF Thang protein phan tCr luring thap; Mau chu chat; Phan Joan sau qua cat; Phan doan tai can bang cat; Phan doan rera % ddB; Phan doan dung ly 20 % ddB; Phan doan dung ly 100 % ddB 20,1 kDa cua thang phan tCr lucmg, tucmg Ong veri KAt qua din di SDS-PAGE cho thay a cac kich thirac dm protein hGM-CSF ly thuyet pH gieng 2, 3, 4, la cac phan doan protein dung djch can bang (pH,5) stn barn cna hGM-CSF Wang ing vai phan doan protein tang sit, tit chu vao chit trao dai anion cat Q Sepharose FF gAn chat, Olin doan protein sau qua cat, phan nhu dien hoan town khong co sv xuat hien doan tai can bang cat va phan doan nfa, chnng raj cda protein phan doan dung dich sau titian thay milli phan doan chu chat c6 su xuat qua cat (Hinh 5A, gitng 3), mac du co sv raa hien mat vach protein nAm gifta vach 14,4 kDa va Trang 17 Science & Technology Development, Vol 17, No.T4-2014 troi met phan hGM-CSF a giai down tai can bang cat (Hinh 5A, gieng 4) d phan doan dung ly veri 20 % ddB, chimg toi they ce su xuAt hien met vach protein dam, có kich thuac Wang Ung vai kich thuac du doan cua hGM-CSF va toan be protein the duoc dung ly tai phan doan (gieng 6) va khong can tai ruing de mad cao hon (Hinh 5A, gieng 7) De khang dinh cac protein tinh sach duoc chinh la hGM-CSF, chang toi tien hanh lai voi khang the die hieu cho hGM-CSF Ket qua lai trinh bay Hinh 5B cho they cac protein chitng toi thu duoc qua qua trinh tinh sach bang sic ky trao dei anion chinh la protein mix tieu hGM-CSF Ngoai ra, mire tinh sach cua protein thu nhan duoc danh gin bang phan mem xac dinh dam de va ket qua cho they de tinh sach cua hGM-CSF thu duoc sau qua trinh sic kY trao dei anion la 97,4 % (ket qua khong trinh bay) Thir nghiem Wang ti,r Arai phucrng phap dung ly bang pH, ket qua cho they bang each giam pH kitting cho phep chimg toi thu duoc protein hGMCSF tinh sach (lc& qua khong trinh bay a day) Nhu vay, tir nhftng ket qua ten chimg toi ket luAn da tinh sach ding protein hGM-CSF chu chat to bao E colt BL21(DE3)/pET-hGM bang sac kV trao dei anion sir dung cot Q Sepharose FF Arai phucmg phap dung ly tang neng de mu6i KET LUAN Nhu v4y, chUng toi da too clang tong Bong to bao E coil BL21(DE3) co mang vector tai to hop pET-hGM DOng dr kha nang bleu hien protein hGM-CSF chu chat to bao E coll Su bieu hien hGM-CSF da throe kiem chirng bang phuong phap SDS-PAGEva khang dinh lei bang phucrng phap Western blot Protein duoc hen hinh tinh sach bang sac kV trao dei anion sir dung cot Q Sepharose FF vai phircrng phap dung ly tang ruing de muei Mire da tinh sach cila protein duoc xac dinh bang phan mem dinh dam de voi ket qua dat 97,4 % Chung co the duoc sir dung lam ging de cung a/3 nguen nguyen lieu hGM-CSF cho quy trinh sin xuAt hGM-CSF tai to hop ang dung y hoc a Viet Nam Cloning, periplasmic expression and purification of hGM-CSF (Human Granulocyte - Macrophage ColonyStimulating Factor) in Escherichia coli • • • Phu Sang Nguyen Thanh Thao Nguyen Hieu Tran Van University of Science, VNU-HCM SUMMARY • Human GM-CSF is a cytokine consisting of 127 amino acid residues, with four cysteines being involved in two disulfide bonds Although GM-CSF is glycosylated in Trang 18 its natural form, the glycosylation perhaps has not been involved in its biological function GM-CSF stimulates the survival, proliferation, and differentiation of hem at TAP CHI PHAT THIEM MN, TAP 17, Si 14-2014 transformants were induced by IPTG and opoietic progenitor cells and also enhances examined for hGM-CSF production the functional properties of mature myeloid Periplasmic proteins were released by cells GM-CSF is used as a therapeutic osmotic shock treatment The expression of agent in various clinical cases such as recombinant hGM-CSF was evaluated by neutropenia following chemotherapy, bone SDS—PAGE in total, cytoplasmic and marrow transplantation, acute myeloid periplasmic fractions The recombinant hGMleukemia In this study, we report the CSF in periplasmic fraction was then results on the cloning and expression of subjected to ion exchange chromatography recombinant human GM-CSF in the using Q Sepharose FF column with saltperiplasmic space of Escherichia coll The increment elution step SDS-PAGE showed hGM-CSF gene was amplified by there was a visible expression of polymerase chain reaction using two recombinant hGM-CSF in the periplasmic oligonucleotide primers containing Ncol and fraction of the E coli BL21(DE3)/pET-hGM Xhol restriction sites This DNA fragment and a purified band with the purity of 97.4% was successfully cloned between the Ncol after ion exchange chromatography This and Xho I sites of the plasmid pET-22b, in result was further confirmed by Western blot frame with the pelB signal peptide sequence using anti-hGM-CSF antibody The expression vector pET-hGM was transformed into E coli BL21(DE3) and the Keywords: hGM-CSF, periplamic, anion exchange chromatography TAI LIEU THAM KHAO [s] G Marini, et al., Recombinant human granulocyte - macrophage colony-stimulating factor: effect of glycosylation on Electronic pharmacokinetic parameters Journal of Biotechnology, 10, 271-278, (2007) [2] M.R Walter, et al., Three-dimensional structure of recombinant human granulocyte macrophage colony-stimulating factor J Mo! Biol, 224, 1075-85 (1992) [3] J.O Armitage, Emerging applications of recombinant human granulocyte -macrophage colony - stimulating factor Blood, 92(12), 4491-4508 (1998) [4] P.K.B Ghosh, D Karnik, Human granulocyte - macrophage colony stimulating factor: The protein and its current & emerging of Journal Indian applications, Biotechnology, 6, 435-448 (2007) [5] J Messens, J.F Collet, Pathways of disulfide bond formation in Escherichia coli Int J Biochem Cell Biol, 38, 7, 1050-62 (2006) [6] R Greenberg, et al., Expression of human mature active, biologically granulocyte - macrophage colony stimulating factor with an E coli secretory expression system Curr Microbiol, 17, 6, 321-332 (1988) [7] J Sambrook, D.W.R., Molecular Cloning: A laboratory manual, third edition cold spring Harbor laboratory (2001) [8] F.M Ausubel, Brent, R Kingston, R E Moore, D D Seidman, J G Smith, J A & K Struhl, Current protocols in Molecular Biology, New York: Wiley (1989) Trang 19 ... ding protein hGM- CSF chu chit to bao E coil BL21(DE3)/pET -hGM Tinh such hGM- CSF chu chit to bio E coil BL21(DE3)/pET -hGM bing sic kI trao di anion Do protein hGM- CSF co pI lchoang 5,5 la mat protein. .. theo phtrong phap SDS-kiem theo mo to cita Sambrook va Russell [7] Thiet kg plasmid tai to Ito plET -hGM mang gen hgm- csf ml boa cho protein hGM- CSF Gen hgm- csf ma hea cho protein hGM- CSF duqc... hGM- CSF (Human Granulocyte - Macrophage ColonyStimulating Factor) in Escherichia coli • • • Phu Sang Nguyen Thanh Thao Nguyen Hieu Tran Van University of Science, VNU-HCM SUMMARY • Human GM-CSF