cd NA ,, Expression and Character iza tion of Type-1 Pla sm inogen Activa tor Inh ibitor in P ich ia p astoris PA I21 , 1% 7 d, W estern b lo t

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1 14 5 V o l114,n o Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy O cṫ 1998 g cd NA P p astoris 3 (, ) 379 PA I21 cdna AOX1 PHO 1, py IS21 P p astoris, H is + M u t -,, 1% 7 d, SD S2PA GE, PA I21 W estern b lo t 43 51kD 4 PA I21 PA I21, 26%, 4 m ggl ; AU gm g PA I21 :, g,, Expression and Character iza tion of Type-1 Pla sm inogen Activa tor Inh ibitor in P ich ia p astoris H e Cheng T ang H u i2b in T ian Yu Song Hou2Yan Zhu Yun2Song 3 (D ep artm ent of M olecular Genetics, S hanghai M ed ical U niversity, S hanghai ) Abstract By in serting PA I21 cdna coding 379 am ino acid residues in to the m ethylo troph ic yeast exp ression vecto r ph IL 2S1 con tain ing AOX1 p rom o to r and the sequences of PHO 1 se2 creting signal pep tides, the exp ression p lasm id py IS21 w as con structed and then tran sfo rm ed in to P p astoris GS115 T he H is + M u t - tran sfo rm an ts w ere selected and ferm en tated in flask s w ith low den sity A fter 7 days of 1% m ethano l induction, the exp ression m edia w ere analyzed and the exp ressed p ro tein w as verified to be recom b inan t hum an PA I21 (rh PA I21) in differ2 en t m o lecu lar w eigh ts of kd by SD S2PA GE, b ioactivity and W estern b lo t detection T he exp ressed rhpa I21 cou ld be effectively secreted in to m edia, and its h ighest quan tity w as estim ated to be 26% of to tal p ro tein s o r 4 m ggl in m edia T he specific activity of rhpa I21 w as AU gm g T he non2hom ogeneity of M r of exp ressed rhpa I21 m ay be related to the length of its o ligo saccharide chain ṡ T he PA I21 exp ressed in P p astoris w as very helpfu l to fu rther study its structu re and physio logical function Key words: M ethylo troph ic yeast, T ype21 p lasm inogen activato r inh ib ito r, Exp ression, O ligo saccharide chain 3 : , : China Academic Journal Electronic Publishing House All rights reserved

2 g 2 (p lasm inogen activato r inh ib ito r2g, PA I21) 53kD, SER P IN PA I2g 3 (t2pa ) (u2pa ) [ 1, ] PA I2g [ 2, ] H epg2 PA I2g [ 3 ], PA I2g PA I2g u2pa, u2pa [ 4 ] PA I2g, PA I2g PA I2g,, PA I2g [ 5, 6 ], PA I2g [ 7, 8 ],, PA I2g, (AOX) PA I2g, P ich ia p astoris PA I2g PA I21 cdna pyzhb22 PA I21 PA I21 JM 83 ph IL 2S1 GS115 Invitrogen DNA T 4 DNA G IBCOBRL D 2 Serva YNB (W go ) D ifico, 112 DNA M an iatis [ 9 ] 113 P p astoris GS m o lgl L ic l Bg1g PA I21 pys I21 ph IL 2S1 20 Λg (20 Λl), 10 Λg DNA 011 m l GS m in, 017 m l 40% PEG4000gT E2L ic l,, 30 m in 37 5 m in, 011m l, M D (1134% YNB, %, 1% D ex tro se, 115% agar), d, 114 H is + M u t - MM (1134% YNB, %, 015%, 115% A gar) M D, MM M D 115 H is + M u t - 4 py IS21 ( ) 2 ph IL 2 Sl (31 36 ) 50 m l BM Y (011 m o lgl ph 610, 1134% YNB, %, 1% ), 30 A 600nm 410, 10 m l BMM China Academic Journal Electronic Publishing House All rights reserved

3 (011 m o lgl ph 610, 1134% YNB, %, 1% ), 1 m l, 100% 1%, 7 d, - 30, SD S2PA GE : SD S2PA GE, [10 ], PA I21 ; PA I21, PA I PA I21 (W estern b lo t ) : [11 ] 117, G2250 [ 12 ] SD S2PA GE,, (CS2910) 500 nm, 600 nm, PA I PA I-1 py IS-1 pyzhb22 PA I21 cdna A T G PA I TA G 66 bp 3, EcoR g Bam H g, 112 kb PA I21 cdna ph IL 2Sl EcoR g Bam H g, PA I21 cdna PHO 1 22, (AOX), PA I21 py IS21 ( F ig 1 ) EcoR g + Bam H g Bg1g + Salg Salg, py IS kb 813 kb ; 1176 kb 2137 kb 2144 kb 219 kb 2137 kb 7112 kb (F ig 2) 212 PA I-1 P p astoris 20 Λg GS115, 120 6gΛg DNA MM M D, Bg1 g py IS21 GS115 H is + M u t - 7, 6% 4 ( ) 2 (31 36 ), 7 d, SD S2PA GE, F ig 1 Construction of the yeast exp ression p lasm id of PA I China Academic Journal Electronic Publishing House All rights reserved

4 55 kd 4 (F ig 3), 26%, 1d,,,,, 6 d ( ) F ig 2 A garo se gel electropho retic patterns of frag2 m ents of py IS21 digested by restriction endonucleases M : Κ- DNA gh ind g standard mo lecular m arkers A : py IS- gbamh g + EcoR g : 112 kb, 813 kb B: py IS- 1gBgl g + Sal g : 1176 kb, 2137 kb, 2144 kb, 219 kb C: py IS- 1gSal g : 2137 kb, 7112 kb F ig 3 SD S2PA GE analysis of PA l21 exp ressed in P p as2 toris exp ressed in flasks Stained w ith silver salts D, H:M o lecular m ass m arker, indicated in kd A, B, C, E: Exp ression m edia of N o 6, 18, 24, 30 strains harbo ring the p lasm id py IS21 F, G: Exp ression m edia of N o 31 and 36 strains harbo ring vecto r p lasm id ph IL 2S1, as negative contro ls : SD S2PA GE, kd, PA I2g :, 4 50 kd t2pa, (F ig 4) ;, 30 7 d PA I AU gm l, PA I Λggm l, AU gm g : W estern b lo t, kd PA I21, PA I21 F ig 4 Reverse fibrin2agaro se zymography analysis of inh ibito ry activity of PA I21 A fter 7 days of m ethano l induction, the m edia w ere app lied to SD S2PA GE and then stick to fibrin2agaro se p late A, B, C, D: Exp ression m edia of N o 6, 18, 24 and 30 strains E: Exp ression m edia of N o 31 strain, as a negative contro l T he blurring band show n by an arrow indicates the activity of PA I China Academic Journal Electronic Publishing House All rights reserved

5 (F ig 5) F ig 5 W estern blat analysis of PA I21 exp ressed in P p ostoris A : Purified PA I21 exp ressed in E coli B, E: Exp ression m edia of N o 31 and 36 strains, as negative contro ls C, D: Exp ression m edia of N o 24 and 30 strains 3 GS115 P 1p astoris, (AOX1, AOX2) [ 13 ], 2,, AOX1 (PAOX1), AOX1, AOX2, PAOX1 P p astoris,, [ 14 ], PA I21 cdna PAOX1, PHO 1, PA I21 py IS21, Bgl g, PA I21 cdna AOX1 3,, AOX1, AOX1, ( AOX2 ), MM, M D H is + M u t - ( h is ) PA I21 cdna,, P p astoris, [ 15 N 2 ], PA I kD,, T schopp [ 16 ] P p astoris PA I21 P p astoris,,,,, ph, PA I21,,,, References 1 B iemond B J,L evim, Co ronel R, Janse M J, ten2cate J W, Pannekoek H T h rom bo lysis and reocclusion in experim ental jugular vien and co ronary artery throm bo sis Effects of a p lasm inogen activato r inhibito r type 12neutralizing monoclonal antibody C ircu lation, 1995, 91 (4) : ,,,,, 1 PA I21 1 (Sun You2H ua, Zhang X in2p ing, D ing H ao, Zha X i2l iang, Song Hou2Yan, Zhu Yun2Song Comparative studies on China Academic Journal Electronic Publishing House All rights reserved

6 the functions and characteristics of the glyco sylated, the nonglyco sylated and the recom binant PA I21, A cta B iochim B io2 p hy s S in), 1996, 28 (2) : ,,,,, 1 H epg2 PA I211 (D ing H ao, W u X iao2l i, Sun You2H ua, Zhou X ing2h ua, Zhu Yun2Song, Song Hou2Yan Purification of PA I21 secreted from H epg2 cell by imm unoaffinity ch rom atography Ch in B iochem J ), 1994, 10 (1) : Soff G A, Sanderow itz J, Gately S, V errusio E,W eiss I, B rem S, Kw aan H C Exp ression of p lasm inogen activato r in2 h ibito r type 1 by hum an p ro state carcinom a cells inh ibits p rim ary tumo r grow th, tumo r2associated angiogenesis, and m etastasis to lung and liver in an athym ic mouse model J C lin Invest, 1995, 96 (6) : ,,,,, 1I cdna 1 : 1 1 : (H e Cheng, H e P ing, Zhang X in2p ing, H ao Q in, Song Hou2 Yan, Zhu Yun2Song T he cloning and exp ression of p lasm inogen activato r inh ibito r21 (PA I21) cdna in E scherich ia coli Edito rial Board of Chinese Journal of B io techno logy ed Collected p ap ers in B iotechnology Beijing: Chem ical Industry P ress), 1994: ,,,, 1 PA I21 1 (H e P ing, H e Cheng, Zhang H ua, Song Hou2Yan, Zhu Yun2Song Purification of recom binant hum an PA I21 p roduced in E coli Ch in B iochem J ), 1996, 12: (6) ,,,,,, 1g cdna 1 (Zhang X in2p ing, H ao Q in, Sun You2H ua, Chen W ei, Chen You2M ei, Song Hou2 Yan, Zhu Yun2Song Construction of p lasm inogen activato r inhibito r21 cdna clone and exp ression in m amm alian cells A cta A cad M ed S hang hai), 1996, 23 (3) : Gardell S J, H are T R, H an J H,M arkus H Z, Keech B J, Carty C E, E llis R W, Schultz2L D Purification and characteri2 zation of hum an p lasm inogen activato r inh ibito r type I exp ressed in Saccharom yces cerevisiae A rch B iochem B iop hy s, 1990, 278 (2) : Sam brook J, F ritsch E F,M aniatis T M olecu lar C loning: a laboratory m anual, 2nd ed Co ld Sp ringer H arbo r: T he Co ld Sp ring H arbo r L abo rato ry P ress, 1989: Boo th N A,M acgrego r I R, H unter N R, Bennett B P lasm inogen activato r inh ibito r from hum an endo thelial cells Purifi2 cation and partial characterization E u r J B iochem, 1987, 165 (3) ,,, 1 1 (Zhu Yun2Song, Gu Yin2L iang, Zhu Zheng2M ei, Gu T ian2jue A new electropho resis technique: p ro tein transfering elec2 tropho resis P rog B iochem B iop hy ), 1984, 60 (6) : : (Xu X iu2l an ed E xp erim ents and D irections of B io2 chem istry Beijing: Ch inese M edical Science and T echno logy P ress), C regg J M,M adden K R, Barringer K J, T h ill G P, Stillm an C A Functional characterization of the two alcoho l oxidase genes from the yeast P ich ia pasto ris M ol Cell B iol, 1989, 9 (3) : Faber K N, H arder W, A b G, V eenhuis M Review: m ethylo troph ic yeasts as facto ries fo r the p roduction of fo reign p ro2 teins Y east, 1995, 11 (14) : C rinna L S; T schopp J F Size distribution and general structural features of N 2linked o ligo saccharides from the m ethy2 lo troph ic yeast, P ich ia pasto ris Y east, 1989, 5 (2) : T schopp J F, Sverlow G, Ko sson R, C raig W, C rinna L H igh2level secretion of glyco sylated invertase in the m ethy2 lo troph ic yeast, P ich ia p astoris B iogt echnology, 1987, 5 (12) : China Academic Journal Electronic Publishing House All rights reserved