Preparation and Characterization of a Novel Recombinant Imaging Agent Directing Thrombus

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 17 (6) : ,,,, (, ) 3 2 GMP2140 SZ51, 1 SZ51 (ScFv2SZ51) 1 (MT2 ) cdna, BL21 (DE3) plyss, HLMT 40 mgπl 84 %, Q2 Sepharose FF Sephadex G250 SDS2PAGE 95 % HPLC HLMT , p I 310 Western,HLMT 2 GMP2140 (AAS),HLMT HLMT,,, TQ92,R445 Preparation and Characterization of a Novel Recombinant Imaging Agent Directing Thrombus ZHOU Bo, SUN Yan, CHEN Yu, YU Mei2min, RU Bing2gen 3 ( National Laboratory of Protein Engineering, College of Life Sciences, Peking University, Beijing , China) Abstract A novel recombinant imaging protein directing thrombus was developed as potential thrombus detec2 tion agent A recombinant gene was constructed with one mole of SZ51 single2chain fragments of variable re2 gion (ScFv), which reacted specifically to the 2granule membrane protein GMP2140 on the surface of activated platelets during thrombus forming, and one mole of MT which was fused to C2terminus as labeling moiety It was expressed in E coli BL21 (DE3) plyss The recombinant products named HLMT were accumulated as cy2 toplasmic inclusion bodies and then refolded in vitro Several factors influencing the refolding activity were ex2 amined and a way of efficient recovery of active protein was established The refolding solution was applied to Q2Sepharose FF ion exchange chromatography and Sephadex G250 chromatography The purity determined by SDS2PAGE was over 95 % The homogeneity of the purified protein was proved by HPLC Mass spectrometry showed that the molecular weight of HLMT was The p I was 310 The atomic absorption spectra2pho2 tometry (AAS) proved that the HLMT had the same metal binding capacity as metallothionein Western blot2 : , : (No 962C02201) 3 Tel : ,Fax : ,E2mail edu cn,,1972 3, Received :January 15,2001 ;Accepted :April 18,2001 Supported by the 9th Five Years Plan Special Research Programs of China, (No 962C02201) 3 Corresponding author Tel : ,Fax : ,E2mail edu cn China Academic Journal Electronic Publishing House All rights reserved

2 ting showed that the HLMT had the activity of anti2human activated platelets as the intact antibody SZ51 HLMT was expected to be a fast, effective targeting and imaging agent in the clinical thrombus detection Key words imaging agent directing thrombus, metallothionein, ScFv, isolation and purification, character2 ization,, ( Shimadou Dual2wavelength TLC SCANNER CS2910) ; (DVT) ( PE), HPLC ( HP G2000SW) ; ( Hewlett, Packard G2025, LD2TOF system) ; PU9200 (Philips ),, 112 [4 ] 11211, pet25 2ScFv2MT E coli BL21, (DE3) plyss,, ml Amp (100 mgπl) LB,, 1 10,, IPTG (, 011 mmolπl), 4 h, [5 ] (metallothionein, MT) [1, ] MT,, 011 % Tri2, tonx mmolπl Tris2HCl (ph 810), 3, 50 mmolπl Tris2HCl,, rπmin 10 min, [2 ] GMP140 SDS2PAGE 50 mmolπl Tris2HCl, 1 mgπml SZ51 Fab, 60 min, (DOC) 015 %, 30 min, rπmin 10 min, GMP140 [3 ] [4 ], SDS2PAGE 1 SZ51 (Shimadou Dual2wavelength TLC SCANNER CS2910) (ScFv2SZ51) 1 MT2 cdna, pet5a2scfv2mt, BL21 (DE3) plyss (8 molπl,50 mmolπl 2 ),4, 24 h, h p ET5a2ScFv2MT BL21 (DE3) plyss 24 h [6 ] ( ) ;SDS2PAGE BIO2RAD rπmin 15 min, 50 mmolπl Tris2HCl (ph ;Q2Sepharose Fast Flow Sephadex G ), Q2 Pharmacia2LKB Biotech Co ; Sepharose Fast Flow (210 cm 12 cm), 0 (Pharmacia ) ; (BIO2RAD) ; (50 mmolπl Tris2HCl, 215 molπl, 5 mmolπl EDTA, 1125 mmolπl GSH, 0125 mmolπl GSSG,1010 mmolπl NaCl, ph 910), 50 1,4, 110 molπl NaCl, 60 mlπh, UV240 ( ) ;, Sephadex G250 (310 cm China Academic Journal Electronic Publishing House All rights reserved

6 : 783 100 cm), ph 810,50 mmolπl Tris2HCl, 6 mlπh,, SDS2PAGE, [7 ] 11215 HPLC Varian CDS 401 Liquid Chromatography,715 mm 300 mm( ID L) 10 l, ph 810,50 mmolπl Tris2HCl, 1 mlπmin 11216 Hewlett

3 6 : cm), ph 810,50 mmolπl Tris2HCl, 6 mlπh,, SDS2PAGE, [7 ] HPLC Varian CDS 401 Liquid Chromatography,715 mm 300 mm( ID L) 10 l, ph 810,50 mmolπl Tris2HCl, 1 mlπmin Hewlett Packard G2025, LD2TOF system [7 ] (a) [7 : ] (b) (AAS) [8,9 ] :, 115 ml, nm, 012 nm, 8 ma,znso 4 Zn Western MT, (NC), MT, HRP IgG, GMP140, Western [10 ] SDS2PAGE, Fig 1 SDS2PAGE of the recombinant protein during the prepara2 tion process 1 :Purified recombinant protein HLMT; 2 :Recombinant protein after renaturation ; 3 : Inclusion body after lysozyme and DOC treatment ; 4 : Supernatant after centrifugation ; 5 : Inclusion body before lysozyme and DOC treatment ; 6 :Total proteins of E coli cells pro2 ducing HLMT; 7 :Molecular mass marker Fig 2 Q2Sepharose Fast Flow chromatography of HLMT The column was equilibrated with 50 mmolπl Tris2HCl, ph 810 The bound proteins were eluted with NaCl gradient (0 110 molπl) in 50 mmol Tris2HCl, ph 810 The elution rate was 60 mlπh Peak 2 indicates the desired recombinant protein 38 kd ( Fig11) (Fig 4),, 24 %,38 kd, 214, HLMT , DOC 38430, [ DOC ], ph,,hlmt p I 310, 84 % Fig 2 Fig Zn 2 + Table HPLC 7 HPLC, Zn China Academic Journal Electronic Publishing House All rights reserved

784 17 Fig 3 Sephadex G250 chromatography of HLMT The curve indicates size exclusion chromatography moni2 tored by A 280 The elution buffer was 50 mmolπl Tris2HCl, ph 810, with flow rate of 30 mlπh

4 Fig 3 Sephadex G250 chromatography of HLMT The curve indicates size exclusion chromatography moni2 tored by A 280 The elution buffer was 50 mmolπl Tris2HCl, ph 810, with flow rate of 30 mlπh Peak 1 indicaes the desired recombinant protein The curve 222 indicates the peak of AAS(Zn) during the course of size exclusion chromatography Fig 4 HPLC of the purified HLMT, Table 1 The metal binding capacity of HLMT c (protein)πmmol L - 1 The amount of Zn 2 + conjugated by c(zn 2 + )Πmmol L - 1 one protein molecule (theoretical) The amount of Zn 2 + conjugated by one protein molecule (experimental) Recombinant protein SZ Π Π Π 217 Western 3 Western, SDS2PAGE, GMP140, 311, SZ51, Fig15 Fig 5 Western blotting of HLMT 1 :SDS2PAGE of platelet antigen activated by thrombin ; 2 : Arrow indicates the positive interaction between recombinant protein and GMP140 of activated platelet,, DNA RNA, DOC, DOC, 84 %,,,,, [11 ],, China Academic Journal Electronic Publishing House All rights reserved

5 6 : , ph,, [12 ], ph, HLMT ph,,,,,, 312,, SDS2PAGE 95 % HPLC Sephadex G250,, 7104 Zn 2 +, 4 3,,, 30 min, 313 Western, GMP2140,,,, ( References) clusion bodies overexpressed in Escherichia coli by controlled introduc2 1,,, ( Ru Bing2gen, Pan Ai2hua, Huang Bing2qian, Zhang Long2xiang Metallothionein Prog Biochem Biophys),1991,18(4) :254 2 Sawyer J R,Tuchker P W and Blattner F R Metal2binding chimeric an2 tibodies expressed in Escherichia coli Proc Natl Acad Sci USA,1992, 89 : ,,,Lefranc M P, SZ51 Fab E coli ( Gu Jian2ming,Zhang Xiao2min,Xia Li2jun,Liu Yue,Lefranc M P, Ruan Chang2geng Expression of the anti2human activated Platelet anti2 body SZ51 chimeric Fab fragment Chin J Hemotol) 1995,16 (9) : ,, SZ51 BL21 (DE3) plyss (Sun Yan,Zhang Pei2chuan, Ru Bing2gen Study on the ex2 pression of a kind of metallothionein and SZ51 recombinant protein in the E Coli BL21 (DE3) plyss Strain Chin J Biochem Mol Biol),1999,15 (3) : ,, PH085 ( Zhong Hua2lin,Li Bo2 liang, Ao Shi2zhou Cloning and highly efficient expression of yeast PH085 gene in E coli Acta Biochim Biophs Sin), 1992,24 : Hua Zi2chun,Dong Chen,Zhu De2xu Renaturation of Recombinant Hu2 man Pro2Urokinase Expressed in Escherichia coli Biochem Biophys Res Commun,1996,220 : ,, ( 2 ) : ( Zhang Long2xiang, Zhang Ting2fang,Li Ling2yuan Experimental Methods and Techniques in Biochemistry 2nd ed Beijing : Higher Education Press),1997 : , ,,,, ( Pheretima guillelmi) (Li Ling2yuan,Ma Hong2bao,Lu Ying2chun,Ru Bing2gen, Shen Tong Isolation, purification and biochemical properties of metallothio2 nein from earthworm( Pheretima guillelmi) Chin Biochem J ),1994,10 (4) : ,,, (Metallo2 thionein) ( Ru Bing2gen,Pan Ai2 hua, Wang Zheng2xin, Zhang Long2xiang Isolation, purification and identification of metallothionein from mouse liver Chin Biochem J ), 1991,7(3) : : (Lu Sheng2dong Experimental Techniques in Modern Molecular Biolo2 gy Beijing : Higher Education Press),1993 : : 1999 : (Marshak D R, Kadonaga J T,Burgess R R et al Strategies for Protein Purification and Characterization : A Laboratory Course Mannual Cold Spring Harbor Labarotory Press 1996) 12 Tsumotok, Shinoki K, Kondo H, Uchikawa M, Juji T, Kumagai I Highly efficient recovery of functional single2chain Fv fragments from in2 tion of oxidizing reagent2application to a human single2chain Fv frag2 ment J Immunol Methods,1998,219( 122) : China Academic Journal Electronic Publishing House All rights reserved

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