ISSN 100727626 CN 1123870gQ 16 4 Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 4, 452 457 2000 8 NAD H- b5 (, 350025) NADH 2 b5, (b5r ), (C203Y) b5r cdna pgex22t, BL 21 W estern GST 2b5R 2Sepharo se 4B, GST 2b5R GST 2b5RC203Y GST 2b5R GST 2b5RC203Y,,,, C203Y NADH 2 b5,, GST, Q 78 Expression and Pur if ica tion of W ild and M utan t Type NAD H-Cytochrom e b5 Reducta se in E. coli W AN G Yao,W U Yu2shu i, LAN Feng2hua, TAN G Yu2cha i, ZHU Zhong2yong (Centre f or L aboratory M ed icine, F uz hou General H osp ital, F uz hou 350025, Ch ina) Abstract To characterize the effect of C203Y m issen se m u tation on the enzym e function of NADH 2cytoch rom e b5 reductase, C203Y m u tan t b5r cdna w as iso lated from p lasm id pw R 24502 b5rc203y by RCR. T he w ild type and m u tan t b5r cdna s w ere cloned in to the exp ression vecto r pgex22t. T he exp ression s of GST 2fu sed w ild type and C203Y b5r m u tan t p ro tein w ere analyzed by SD S2PA GE and W estern b lo tting after induced by IPT G fo r 4 hou rṡ T hen GST 2fu sed w ild type b5r and GST 2fu sed C203Y b5r w ere purified w ith single2step affin ity ch rom atograph ic co lum n of Glu tath ione Sepharo se 4B. T he purity of bo th b5r p ro tein s w as analyzed by electropho resis on a PA GE and a single band w ith a m o lecu lar m ass of 58 kd w as ob tained. Com pared GST 2fu sed b5r w ith GST 2fu sed b5rc203y exp ressing in E. coli, it w as found that m u tan t enzym e activity w as the sam e as w ild enzym e activity, bu t less therm o stab ility and a greater su scep tib ility to tryp sin than the no rm al coun terparṫ T he resu lts suggest that th is am ino acid sub stitu tion influences secondary structu re of the enzym e, resu lting in enzym e in stab ility. Key words NADH 2cytoch rom e b5 reductase, P rokaryo tic exp ression, GST 2fu sion p ro tein, A ffin ity ch rom atography (RCM ), (L eu72p ro,, NADH 2 b5 Cys203T ry) [ 2, 3 ] b5r RCM [ 1 (b5r ) ], b5r,,, 5 9, :,, 1963 7,, T el: (0591) 372129, Fax: (0591) 3732129 : 1999209203, : 1999212229
4 : NADH 2 b5 453, pgst 2b5R pgest 22T pgst 2b5RC203Y,, b5r cdna R sag 10U, GST 2b5R GST 2b5RC203Y, b5r 1 111 11111 : B L 21, pgex2 2T Pharm acia 11112 : E cor g B am H g, R sag, T 4 DNA Ferm en tas ; T aq DNA 5 174DNA gh ae g P rom ega W estern Zym ed L abo rato ries [ 4, b5r ] ; GST Pharm acia 11113 : PE2400 DNA (PE ) ; 373 DNA (AB I ) ; W estern (B io2r ad ) 112 11211 b5r cdna, : T 1 5 2GGGGA TCCT TCCA GCGCTCC A CGCC23, 5 B am H g ; T 2 5 2GGGGAA T TCCCCTCA GAA GA CGAA GCA 23, 5 E cor g 846 bp, b5r 275, 11212 pgst 2b5R pw R 4502b5R cdna pw R 4502 b5r cdna C203Y [3 ]pgst 2b5R F ig. 1 11215 W estern b5r pw R 4502b5R cdna, T 1 T 2 PCR PCR 25 l, PCR 94 45 s, 60 5 s, 72 60 s, 8% (PA GE), 11214 pgst 2b5R IPT G pgst 2b5R pgst 2b5RC203Y, BL 21, 1 mm o lgl IPT G, 100 ggm l LB, 37, 0, 1, 2, 3 h 1 m l,, 2 SD S, 3 m in, 10% SD S2PA GE F ig. 1 Construction of b5r exp ression vecto r in E. coli To construct the exp ression p lasm id of the m utant enzym e, pw R 24502b5R and pgex22t w ere used. The box w ith slants indicates b5r cdna, the filled indicates po sition of the m issense m utation, and the box w ith do ts indicates the GST 2coding region GST 2b5R SD S2 PA GE, 100 V (N C ) b5r 30, 72 10m in DNA, IgG, 42 PCR, E cor g B am H g [ 5 21, ], pgex22t, 11216 GST 2b5R, Pharm acia GST DNA, DNA, : IPT G 11213 b5r cdna (R FL P)
454 16 12 m l, 10 m ggm l 6 l,, 2Sepaharo se 4B,, 50 mm o lgl, GST 2b5R 11217 GST 2b5R GST 2b5RC203Y GST 2b5R GST 2b5RC203Y [ 6 ] GST b5r : 0105 m o lgt ris2 HC l ( ph 7. 5 ), 011 mm o lgl ED TA 2N a2, 0106 mm o lgl DC IP, 0. 03 mm o lgl NADH, 3 m l, 25 (UV 2240 ) 600nm 3 m in, GST 2b5R GST 2b5RC203Y 20 l(1 ggl) 50 mm o lgl T ris2hc l, 1 mm o lgl ED TA, 011 mm o lgl D T T, 10 m in, GST 2b5RC203Y, GST 2b5R GST 2b5RC203Y 3 m o lgl 37, 25, b5r cdna 203 T GC TA C, Cys T ry, b5r cdna F ig. 2 212 b5r cd NA 203 R sa g, b5r cdna R sa g, 321 bp 261 bp 135 bp 74 bp 55 bp, 261 bp 222 bp 135 bp 99 bp 74 bp 55 bp F ig. 3 pgex22t b5r cdna 213 GST-b5R SD S-PAGE F ig. 2 Sequencing of b5r cdna T he arrow idicates the m utation po sition (T GC TA C ) at codon 203 w ith in exon 7 F ig. 3 D iagram of restriction endonuclease analysis 1. M arker; 2. pgst 2b5R; 3. pgst 2b5RC203Y b5r GST 2b5R GST IPT G 2 37, 0 1 2 3 h 1 m l, SD S2PA GE 26 kd GST 57 kd 211 b5r GST 2b5R, b5r cdna E cor g, 4 h ( F ig14), B am H g pgex22t, GST 2b5RC203Y IPT G, 4 h pgst 2b5R pgst 2b5RC203Y F ig. 4 IPT G induced exp ression of GST 2b5R fusion p ro tein in E. coli 1. P ro tein m arker; 225: 57 kd GST 2b5R fusion p ro tein exp ression induced by IPT G at 3, 2, 1 and 0 hours: 628: 26 kd GST exp ression induced by IPT G at 3, 2 and 1 hours
4 : NADH 2 b5 455 214 W estern GST-b5R GST-b5RC 203Y b5r GST 2b5R, 57 kd, GST 217 GST-b5R GST-b5RC203Y, 57 kd GST 2 b5r F ig. 5GST 2b5RC203Y F ig. 7 37 50 215 GST-b5R GST-b5RC203Y 80% 7%,, GST 2 IPT G, b5r 50 10 m in 2Sepharo se 4B, 50%, GST 2b5R GST 2 b5rc03y SD S2PA GE 57 kd 10 m in 20 m in, GST 2b5RC203Y,, 12 m l 52% 18%, 100 200 g F ig. 6 b5r 600 nm 3 m in b5r, b5r 99% GST 2b5R GST 2b5RC203Y 10 m in, GST 2b5RC203Y 3 m o lgl 37, b5r 95% 52% (F ig. 8) F ig. 5 GST 2b5R, GST and GST 2b5RC203Y p ro tein identified by W estern blo tting 1: Recom binant GST 2b5R p ro tein; 2: b5r p ro tein; 3: GST p ro tein contro l; 4: GST 2b5RC203Y p ro tein F ig. 7 H eat stability of the w ild2type and m utant b5r s T he w ild2type and m utant b5r s w ere incubated fo r 10 m in at various temperature w ild type; m utant type F ig. 6 Purification of GST 2b5R and GST 2b5RC203Y fusion p ro tein 1. M arker; 2: GST 2b5R; 3: GST 2b5RC203Y 216 GST-b5R GST-b5RC203Y F ig. 8 T ryp sin suscep tibility of w ild2type and m utant b5r s T he w ild2type and m utant b5r s w ere incubated fo r various tim e at 37 w ith 1. 5 U gl w ild type; m utant type
456 16 3 NADH 2 b5 (b5r; EC 1. 6. 2. [ 7 2) FAD ], : b5r, 50 10, ; m in 20 m in,, P450 b5r, 203, (RCM ), RCM g g [ 8 ] b5r cdna 1974 bp, 903 bp Cys203T ry [ 9 301 ] (References),, 1, RCM g, g, ( ), [ 10 ] b5r, 120 d, g b5r,, RCM g b5r 3,, g [ 11, K catgk m ],,, 80, 8 14,, (A rg57glu L eu72p ro Cys203T ry ), L eu72p ro Cys203T ry Cys203T ry, b5r cdna B am H g E cor g pgex22t, BL 21, GST 2b5R GST 2b5RC203Y IPT G GST 2b5R GST 2b5RC203Y, 2Sepharo se 4B GST 2b5R GST 2b5RC203Y pgex2 2T GST 2b5R GST 2b5RC203Y,, W estern GST 2b5R GST 2b5RC203Y GST 2b5R GST 2b5RC203Y, b5r,,, 1 NADH 2 b5 (H uang Chang2hui, Yu X iu2lin, Zhu Zhong2yong. The studies of NADH 2cytochrom e b5 reductase in patients w ith H ereditary m ethemoglobinem ia. Ch in J H em atol), 1993, 8 (10) : 408 411 2 W u Y S, H uang C H, W ang Y, H uang Q J, Zhu Z Y. Identification of a novel po int m utation (L eu72p ro ) in the NADH 2cytochrom e b5 reductase gene of a patient w ith hereditary m ethemoglobinam ia type İ B ritish J 1998, 102: 575 577 H aem atol, 3,,,,,,, NADH 2 b5 1 (W ang Y,W u Y S, Zheng P Z, L ang F H, Zhu Z Y, T ang Y C, Yang W X, Fang G A. T he identification of po int m utation in NADH 2cytochrom e b5 reducteast gene cdna and gene diagno sis, Ch in J H em atol), 1999, 20 (10) : 321 323 4 L an F H, T ang Y C, H uang C H, Zhu Z Y. E stablishm ents of monoclonal antibodies against hum an erythrocyte NADH 2 cytoch rom e b5 reductase. H y brid om a, 1996, 15: 295 298 5 Sam brook J, F ritsch E F, M aniatis T. M olecu lar C loning, A L aboratory M anual, 2nd ed. N ew Yo rk: Co ld Sp ring H arbo r L abo rato ry P ress, 1989, 9: 888 898 6, 1 NADH 2 b5 1 (Yu X L, Zhu Z Y. P roperties and purification of p ig liver m icro som al NADH 2 cytoch rom e b5. A cta B ioch im B iop hy s S in), 1994, 26 (3) : 297 301 7 Yubisui T, T akesh ita M. Characterization of the purified NADH 2cytoch rom e b5 reductase of hum an eryth rocytes as a FAD containing enzym e. J B iol Chem, 1986, 255: 2454 2457 8 L eroux A, Junien C, Kap lan J2C, Bam berger J: Generalised deficiency of cytochrom e b5 reductase in congenital m ethaemoglobinaem ia w ith m etal retardation. N ature, 1975, 258: 619 625 9 Tom atsu S, Kobayash i Y, Fukum ak i Y, Yubisui T, O rii T, Sakaki Y. The o rganization and the comp lete m ucleo tide
4 : NADH 2 b5 457 sequence of the hum an NADH 2cytochrom e b5 reductase gene. Gene, 1989, 80: 353 361 10 Katsube T, Sakamo to N, kobayash i Y, sek i R, h irano M, T anish im a K, Tomoda A, T akazakura E, Yubisui T, T akesh ita M, Sakak i Y, Fukum ak i Y. Exonic po int m utation in NADH 2 cytochrom e b5 reductase gene of homozygo tes fo r hereditary m ethemoglobinem ia, typeg and type g : Putative m echanism s of tissuedependent enzym e deficiency. A m J H um Genet, 1991, 48: 799 808 11 Sh irabe K, L andim T, takesh ita M, U ziel G, Fedrizzi E, Bo rgese N. A novel po int m utation in a 3 sp lice site of the NADH 2 cytochrom e b5 reductase gene resulta in imm uno logically undetectable enzym e and impaired NADH 2dependent asco rbate regeneration in cultured fibroblasts of a patient w ith type g hereditary m ethemoglobinem ia. A m J hum Genet, 1995, 57: 302 310 SC I2EXPAND ED 2000 7 5, 18 1995 ( In stitu te fo r Scien tific Info rm ation, IS I) (D atabases = SC I EXPAND ED ) (h ttp: ggw o ṡ isiglobalneṫ com gc IW. cgi) 11, ( ) 1 11996, 12 (1) : 104 1081 11991, 7 (4) : 447 4541 11 21,,, ( ) 1 111,, ( ) 1N - 11987, 3 (5) : 468 4721 N IH 3T 3 1 81 31,,,, ( 121,,,, ( ) 1 ) 1 11997, 13 (6) : 699 7031 11 41,,, ( ) 1 FD T P23 2, 3- ) 1g GPA 11998, 14 (5) : 622, 1995, 11 (1) 1114 1161 21 51,, ( ) 1 141,, ( ) 1 pbr 322 DNA DNA C (TH - P ro) 1 DNA 11989, 5 (4) : 335 3381 21 61,,,,,, (,, ) 1 (KDR ) 11998, 14 (1) : 57 611 11995, 11 (6) : 702 705, 11 71,,,, ( 161,,,, ( ) 1 A 2 (PLA 2) ) 1 2 (PA I22) 11989, 5 (5) : 474 4801 11 81,,,,,,, ( ) 1 1 ) 1 E2 11998, 1992, 8 (6) : 755 7581 21 91, ( ) 1 181,,, ( CoA 11996, 12 (2) : 176 ) 1 1811 11 11 101,, ( ) 1 1999, 15 (1) : 105 1081 11 11998, 14 (5) : 512 5171 11 131,, ( 6261 11 1998, 14 (2) : 186 1911 11 151, ( ) 1 11 11998, 14 (5) : 536 5411 21 171,, ( 14 (1) : 15 191 11 11998, 14 (2) : 117 1211 11