- 1. China B iotechnology, 2005, 25 (12) : ( rhop - 1) SDS - PAGE rhop - 1. blot. 21 ( rhop21) 10 g/l, ph

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1 China B iotechnology, 2005, 25 (12) : ( ) - 1 ( rhop - 1) SDS - PAGE rhop %, 8 mol/l,,, 93%,, 98%, 50% W estern blot ( rhop21),, [ 1 ],,, [ 2 ], OP21 [ 3 ], 2004, FDA OP21 PUTTY,, [ 4 ] rhop21, rhop21, rhop rhop21 /pbv221 /E. coli DH5, : : (2003AA2Z3532) 33,: zhaotianna@ sina. com LB : 10 g/l, 5 g/l, 10 g/l, ph B ioflo5000 (NBS ) ; B IOLOGIC DUOFLOW (B io2rad) ; JY922II ( ) ; Power PAC3000 ( B io2rad ) ; A llegra TM 64R ( BECKMAN2COULTER ) ; Sartorius ; Ph211 M icrop rocessor ph Meter ph ( HANNA ) SP2Sepharose Fast Flow, Sephacryl S2100 Fast Flow ( Pharmacia) ; OP21 IgG ( R&D System s, INC), 1. 3 rho P21 E. coli pbv221 rhop21 20 m l 0. 1 mg/m l LB, r/m in500 m l 0. 1 mg/ m l LB ( 1% ), r/m in 8 L OD LB 30%, OD 600

2 2005, 25 (12) : , 8h r/m in 15 m in m l/g ( 20 mmol/l Na2 PO 4, 10 mmol/l EDTA, 1% Tween220, ph6. 0 ),( 400w, 10 s, 15 s, 45 ) r/m in 15m in,, (20 mmol/l Na2PO 4, 1% Triton, ph6. 0),, r/m in, 15 m in ( 20 mmol/l Na2PO 4, 2 mol/l U rea, ph6. 0) 1g/m l 8mol/L (ph6. 0), 4 h r/m in, 20m in, 1. 5 rho P m SP2 Sepharose (XK50 Pharmacia B iotech) 0mol/L, 0. 1mol/L, 0. 2mol/L 1mol/L NaCl ( 8mol/L U rea, 20mmol/L Na2PO 4, ph6. 0 ), SDS2PAGE S2100 ( XK26 Pharmacia B iotech), ( 8mol/L U rea, 20 mmol/l Na2PO 4, ph6. 0),, SDS2PAGE 1. 6 rho P21, 6mol/L, 4mol/L 2mol/L ( 20mmol/L Na2PO 4, ),, Da 1. 7 PAGE (5%, 15% ),, TotolLab, 1. 8 W estern blot SDS2PAGE 5% 4, , 2h PBST 3, ,1. 5 h PBST , SDS2PAGE,35%, SDS2PAGE, 8 mol/l, 60 % ( 1) 1 SDS2PAGE 1: ; 2: ; 3: 8 mol/l ; 4: ;M: marker F ig. 1 Expression, splitting of tha llus and lysis of inclusion bod ies tested by SD S2PAGE L ine 1: Total protein in thallus; 2: The supersanant after sonication; 3: Lysis of inclusion bodies in 8mol/L urea ; 4: The supersanant after centrifugation of the inclusion bodies lysis 2. 2 rho P21 SP2Sepharose 4m l/m in 2(250m l), 150m l,, NaCl 0. 1mol/L NaCl 350m l,0. 2mol/L NaCl 600m l, 1mol/L NaCl 450m l 0. 2mol/L NaCl,, TotalLab 93 % 1mol/L NaCl, ( 2, 3) 2. 3 Sephacryl S2100SP2FF SDS2PAGE SP2Sepharose TotolLab

3 72 China B iotechnology Vol. 25 No SP2Sepharose F ig. 2 Chroma tography of SP2Sepharose 40m l, 2m l/m in,, SDS2PAGE, TotalLab 98%, mg/m l, ( 4, 5) 2. 4 rho P21,, SDS2PAGE, 3 SD S2PAGE SP2Sepharose ( 6) 1: 1; 2: 2; 3: 3; 4: 6; 5: 4; 6: 5;M: Marker, TotalLab, F ig. 3 SD S2PAGE of SP2Sepharose chroma tography Lane 1: Penetration cusp (mark1) 50% ; 2 6: Elution cusplane(mark 2 6) 2. 5 W estern Blot 93% Sephacryl S2100 W estern B lot, 1 15kDa 30kDa 450m l, 2m l/m in,,,, rhop21 ( 7) 4 Sephacryl S2100 F ig. 4 Chroma tography of Sephacryl S2100

4 2005, 25 (12) : SD S2PAGE Sephacryl S2100 1: 1; M: Marker F ig. 5 The protein cusp by Chromatography of molecular exclusion chroma tography tested in SD S2PAGE Lane 1: Elution cusp (mark1) ; 2: Marker 6 RhO P21 1: ; M: Marker F ig. 6 The SD S2PAGE result of rho P21 refold ing 3 L ine1: Refolding p rotein; 2: Marker, rhop21, SDS2PAGE rhop21,, DNA, 8 mol/l, 7 rho P21 1: Marker; 2: rhop21 L ine 1: rhop21 ; line2: rhop21 F ig. 7 Electrophoretogram of rho P21 W estern blot 1: Protein marker 2: rhop21; L ine1: rhop21 dimer; line2: rhop21 monomer SP2Sepharose SP2Sepharose,,, [ 6 ] rhop21 p I 7. 93, ph 6,,, NaCl, NaCl,,,,,, NaCl 10%,, 20%,, 20%, 93%,,,, Sephacryl S2100,,, SP2Sepharose

5 74 China B iotechnology Vol. 25 No Sephacryl S kDa,,, 8 mol/l, ph,,,, TotalLab 98%, 50%,W estern blot, rhop21 rhop21, rhop21 [ 1 ] Haaijman A, D Souza R N, B ronckers A L, et al. BMP27 (BMP27) affects mrna expression of type I, II, X collagen, and matrix Gla p rotein in ossifying long bones in vitro. J Bone M iner Res, 1997, 12 (11) : 1815 [ 2 ] N ishihara A, Fujii M, Kuber S T, et al. Bonemorphogenetic p rotein signaling in articular chondrocyte differentiation. B iochem B iophys Res Commun, 2003, 301 (2) : 617 [ 3 ] Negri A L. Prevention of p rogressive fibrosis in chronic renal diseases: antifibrotic agents. J Nephrol, 2004, 17 (4) : 496 [ 4 ] Vaccaro A R, Patel T, Fischgrund J, et al. A p ilot safety and efficacy study of BMP27 putty ( rhbmp27) as an adjunct to iliac crest autograft in posterolateral lumbar fusions. Eur Sp ine J, 2003, 12 (15) : 495 [ 5 ],.. :, W ang J ZH, Fan M. Protein Technology. Beijing: Science Press, Pur if ica tion and Refold ing of Recom b inan t Human O steogen ic Prote in21 by Ion2exchange Chroma tography and M olecular Exclusion Chroma tography ZHAO Tian2na WANG Shi2li HAN J in2xiang ( Shandong Medicinal B iotechnology Center; Key Laboratory for B iotech2d rugs, M inistry of Health Jinan , China) Abstract Recombinant human O steogenic Protein21 ( rhop21) was generously exp ressed in fermentation. The SDS2PAGE showed that the amount of rhop21 exp ression were 35% in total bacterial p roteins. It was washed and collected after the sonication. 8 mol/l urea was used to dissolve the inclusion body. Most of the impurity were elim inated through the SP2Sepharose ion2exchange chromatography, and the purity rate of target p rotein exceeded 93%. After Sephacryl S2100 molecular exclusion chrom atography, rhop21 was purified to homogeneity. The purified rhop21 was refolded in lower urea gradiently m ethod, and the content of dimer was more than 50%. W estern blot showed that the refolding p rotein was in the form s of monomer or activated dimer. Key words rhop21 Inclusion body Ion2exchange chromatography Molecular exclusion chromatography Refolding