Con struction and B ioactiv ity of Cys57 M utan t of Human In testina l Trefo il Factor
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- Ευτέρπη Βυζάντιος
- 5 χρόνια πριν
- Προβολές:
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1 ISSN CN gQ 16 5 Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 5, (,, ) PCR C 57 h IT F pgex24t 21, IT PG, Glu tath ione2sepharo se 4B, Sephacryl S 100, SD S2PA GE,,,N,, Q 78 Con struction and B ioactiv ity of Cys57 M utan t of Human In testina l Trefo il Factor KOU R u2qin,w AN G W ei,l IL ing2yuan, RU B ing2gen 3 (D ep artm ent of B iochem istry and M olecu lar B iology, Colleg e of L if e S ciences, P ek ing U niversity,n ational L aboratory of P rotein E ng ineering,b eij ing , China) Abstract A m u tan t that deleted Cys57 of hum an in testinal trefo il facto r w as con strucu ted th rough PCR am p lification and exp ressed as fu sion p ro tein w ith GST in E. coli. T he fu sion p ro tein w as purified th rough th ree step s, Glu tath ione2sepharo se 4B affin ity ch rom atography, th rom b in digestion and gel filtration on Sephacryl S 100. T he resu lts of SD S2PA GE, m ass spectrom etry, am ino acid com po sition and sequence of seven am ino acids at the N 2term inu s of the exp ressed p ro tein w ere as expected. T he b io logical activity of the m u tan t w as m uch low er than that of h IT F due to its sen sitivity to pep sin digestion. Key words In testinal trefo il facto r,m u tan t,b ioactivity (in testinal trefo il facto r, IT F) ps2 IT F [ 5 Ch inery ], ps2 IT F [ 1, 2 ] [ 10 3 : T aup in ], (ps2), (SP) IT F 6, IT F 616 kd, Cys12Cys5, Cys22, Cys4, Cys32Cys6 3,, ps2, SP IT F, IT F, 57 [ 3, 4 ], IT F N C SP 3 T el: (010) , Fax: (010) , ps2 IT F, E2m ail: ru@public. easṫ cn. net IT F, 57,, ,, : , :
2 5 : , h, mm o lgl IPT G 4 h, 111 : h IT F cdna (70%, 4, 20 s, pcw T F 15 s), T riton X2100 pb luescrip t KS (+ ) DH 5Α 1%, 30 m in,, pgex24t 21 BL 21 Glu tath ione2sepharo se 4B, (F -, om pt, rb -,m B - ) Pharm acia, 10 U gm g, : E cor g, B am H g, B g l 12 h Sephacryl S2100, g, Kp n g, P st g G IBCO gbrl P rom ega T 4 DNA P rom ega 116 : SD S2 T aq DNA dn T P 2Β2D 2 (SD S2PA GE) [ 9 ] ( IPT G) DNA N [ 9 G IBCO gbrl ] (th rom b in) Sigm a Glu tath ione2sepharo se 4B Sepharcryl2S2 100 Pharm acia h, M ALD ITO F2M S [2 PCR 118 ] : SD PA GE Sp rague2d aw ley (SD ) 2 DNA 211 pgtf- Cys57 : 113 : PCR PCR (DNA 57, T herm al Cycler 480, PER K IN ELM ER CETU S) 5 3, 5 : 5 2GGGGTA CCA G ; (Pharm acia) ; A TCT GA GGTA CGT GGGC; 3 5 2CGGA SD S2PA GE (B io2r ad) ; A T TCCTA TCAAAA GGT T TCT GCT TC, 5 121BM Kp n g B g l g ( (Beckm an) ) ; N 491A h IT F 3 E cor g ( ) h IT F (A pp lied B io system s) ; PCR (D ual2, 200 bp (F ig. 1), w avelength TCL Scanner CS2910) ; 202 bp PCR Kp n g M ALD I2TO F (B ruker) E cor g pb luescrip t KS, pbt F2 Cys57, DNA 114 :, B g l g E cor g pbt F2 Cys57 DNA,, DNA, B am H g E cor g,, DNA,, pcw T FgIT F, PCR : 94 1 m in, s, s, m in PCR Kp n g E cor g pb luescrip t KS,, B g lg E cor g cdna, [7 117 ] IT F IT F2 Cys m o lgl HC l 37 3 F ig. 1 PCR amp lification of target gene 1: 100 bp DNA ladder; 2: T arget gene; 3: contro l B am H g E cor g pgex24t 21,, pgex24t 21, S2 115 : h IT F (glu tath ione S2tran sferase, GST ) 3 [ 6 ] BL 21, 2YT, pgt F2
3 Cys57, F ig. 2 P st g B am H g 1 kb 67 bp (F ig. 3), pgt F2 Cys ITF- Cys57 : IPT G SD S2PA GE, kd (F ig. 4),, 15% 3 4 m ggl F ig. 3 Identification of pgt F2 Cys57 by B am H g (A ) and P st g (B), respectively A : bp ladder; 2. pgt F2h IT F 3. pgt F2 Cys57; B: 1. pgex24t 21; 2. 1 kb ladder; 3. pgt F2 Cys57 F ig. 4 SD S2PA GE of exp ressed fusion p ro tein 1: M o lecular w eigh t m arker; 2. 3: C rude lysate of BL 21 transfo rm ed by pgex24t 21 w ithoutgw ith IPTG induction; 4: C rude lysate of BL 21 by pgtf2 Cys57 w ith IPTG induction F ig. 2 Schem atic diagram of construction of exp ression p lasm id pgtf2 Cys ITF- Cys SD S2PA GE: 18% SD S2PA GE, IT F2 Cys57 (F ig. 5), 616 kd F ig. 5 SD S2PA GE of purified IT F2 Cys57 m utant 1: ITF2 Cys57 m utant; 2: ITF2 Cys57 m utant; 3:M o lecular w eigh t m arker :
4 5 : T ab le 1 6 Table 1 Am ino acid compo sition of m utant p ro tein IT F2 Cys57 iso lated from E. coli cells, Am ino acid Experim ental num bers Theo retical num bers A sx T h r Ser Glx P ro Gly Cys A la V al M et Ile L eu T yr Phe L ys H is T rp ND 1 A rg To tal N o te: V alues of all am ino acids excep t Cys w ere obtained from p ro tein hydro lyzed fo r 24 hours at 110, 6 mo lgl HC l in vacuum. V alue of Cys show ed here w as obtained from p ro tein oxidized w ith perfo rm ic acid F ig. 6 Isoelectric focusing of h IT F and IT F2 Cys57 1: IEF m arker; 2: h IT F; 3: IT F2 Cys57 F ig. 7 W estern2blo t of h IT F2 Cys57 1. h IT F2 Cys57; 2: BSA ; 3: h IT F N : IT F2 214 ITF- Cys57 Cys57 N 7 GlySer GluGluT yrv algly, N (Gly, IT F2 Cys57 Ser) pgex24t 21, Ser IT F2 Cys (F ig. 8B ), : IT F2 Cys , 6619 ( ) h IT F,, [ 7 N 6 ], (F ig. 8A ) IT F2 Cys : ph 5 643,, F ig , IT F ( IT F2 Cys57 h IT F (415) N, ) Cys57,, IT F2 Cys h IT F W estern (F ig. 7),,, 1 g IT F, h IT F2 Cys57,,,, h IT F h IT F,, IT F2,, h IT F Cys57, N
5 F ig. 8 M ass spectrom etry of h IT F and IT F2 Cys57 m utant digested w ith pep sin (A ) IT F; (B) IT F2 Cys IT F2 Cys g, 500 g, g rh IT F rh IT F2 Cys57g, 30 m in 1 m l 016 m o lgl HC l, 5 h, rh IT F2 Cys57 h IT F, rh IT F (F ig. 9 F ig. 10) F ig. 10 U lcer indexes of rh IT F and rh IT F2 Cys57 Contro l group: n = 15; rh IT F group: n = 12; rh IT F2 Cys57 group: n= 12; n: num bers of rat 3 P < 0. 05, 3 3 P < F ig. 9 U lcer grades of rh IT F and rh IT F2 CYS57 Contro l group: n = 15; rh IT F group: n = 12; rh IT F2 Cys57 group: n= 12; n: num bers of rat 3 P < 0. 05, 3 3 P < IT F rh IT F, [ 7,, ] IT F Ch inery IT F, [ 5 ] [ 8 P layfo rd ], IT F, IT F,,,,,,, rh IT F2 Cys57,, IT F,
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