Expression and Pur if ica tion of W ild and M utan t Type NAD H-Cytochrom e b5 Reducta se in E. coli
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- Σέλευκος Παπαϊωάννου
- 5 χρόνια πριν
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1 ISSN CN gQ 16 4 Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 4, NAD H- b5 (, ) 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 , 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 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, :,, ,, T el: (0591) , Fax: (0591) : , :
2 4 : NADH 2 b5 453, pgst 2b5R pgest 22T pgst 2b5RC203Y,, b5r cdna R sag 10U, GST 2b5R GST 2b5RC203Y, b5r : B L 21, pgex2 2T Pharm acia : 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 : PE2400 DNA (PE ) ; 373 DNA (AB I ) ; W estern (B io2r ad ) 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, pgst 2b5R pw R 4502b5R cdna pw R 4502 b5r cdna C203Y [3 ]pgst 2b5R F ig W estern b5r pw R 4502b5R cdna, T 1 T 2 PCR PCR 25 l, PCR s, 60 5 s, s, 8% (PA GE), 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, GST 2b5R, Pharm acia GST DNA, DNA, : IPT G 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.](/docs-images/93/112344538/images/3-0.jpg "5 ), 011 mm o lgl ED TA 2N a2, 0106 mm o lgl DC IP, 0.")
3 m l, 10 m ggm l 6 l,, 2Sepaharo se 4B,, 50 mm o lgl, GST 2b5R 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, 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 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, 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 4 : NADH 2 b 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 GST-b5R GST-b5RC203Y 80% 7%,, GST 2 IPT G, b5r 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%, 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
5 NADH 2 b5 (b5r; EC [ 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 [ ] (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) : 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: 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) : 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: 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: , 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) : 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: 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: 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
6 4 : NADH 2 b5 457 sequence of the hum an NADH 2cytochrom e b5 reductase gene. Gene, 1989, 80: 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: 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: SC I2EXPAND ED , ( 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, ( ) , 12 (1) : , 7 (4) : ,,, ( ) 1 111,, ( ) 1N , 3 (5) : N IH 3T ,,,, ( 121,,,, ( ) 1 ) , 13 (6) : ,,, ( ) 1 FD T P23 2, 3- ) 1g GPA 11998, 14 (5) : 622, 1995, 11 (1) ,, ( ) 1 141,, ( ) 1 pbr 322 DNA DNA C (TH - P ro) 1 DNA 11989, 5 (4) : ,,,,,, (,, ) 1 (KDR ) 11998, 14 (1) : , 11 (6) : , 11 71,,,, ( 161,,,, ( ) 1 A 2 (PLA 2) ) 1 2 (PA I22) 11989, 5 (5) : ,,,,,,, ( ) 1 1 ) 1 E , 1992, 8 (6) : , ( ) 1 181,,, ( CoA 11996, 12 (2) : 176 ) ,, ( ) , 15 (1) : , 14 (5) : ,, ( , 14 (2) : , ( ) , 14 (5) : ,, ( 14 (1) : , 14 (2) :
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A Knowledge M odel for D esign of Population Nutr ien t Index D ynam ics in W heat
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UPTAKE OF CAROTENO ID S BY INTESTINAL M UCOUS CELL S OF LAY ERS AND GOATS IN V ITRO
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STUD IES ON THE GENOTOX IC ITY OF 83- ITS M AJOR M ETABOL ITE 1 HERB IC ID E AND. H eng Zhengchang 1, T O ng 2
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Sensitivity of [Ru(phen) 2 dppz] 2+ Light Switch Emission to Ionic Strength, Temperature, and DNA Sequence and Conformation Andrew W. McKinley, Per Lincoln and Eimer M. Tuite* SUPPLEMENTARY INFORMATION
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ΠΤΥΧΙΑΚΗ ΕΡΓΑΣΙΑ ΕΦΑΡΜΟΓΗ ΤΟΥ ΕΡΓΑΛΕΙΟΥ BALANCED SCORECARD ΣΕ ΙΔΙΩΤΙΚΟ ΝΟΣΟΚΟΜΕΙΟ. Σπουδαστές: Δεληλίγκα Αργυρούλα, ΑΜ: 2008057
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Το άτομο του Υδρογόνου
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Supplemental file 3. All 306 mapped IDs collected by IPA program. Supplemental file 6. The functions and main focused genes in each network.
LIST OF SUPPLEMENTAL FILES Supplemental file 1. Primer sets used for qrt-pcr. Supplemental file 2. All 1305 differentially expressed genes. Supplemental file 3. All 306 mapped IDs collected by IPA program.
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