Angioarrestin( harp1) C FD

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology 23 (2) : Angioarrestin( harp1) C FD,,,,, ( ) 3 Angioarrestin DNA angioarrestin C hfd cdna (MBP) pmal2c 2 2hFD E coli BL21 (DE3), 013 mmolπl IPTG 37 4 h,sds2page, 20 % Western N MBP BalbΠc pet222b ( + ) hfd, ELISA : angioarrestin C hfd, angioarrestin angioarrestin angioarrestin ; MBP ; ; ; Q758 Cloning, Expression and Polyclonal Antibody Preparation of C2terminal FD Domain of Human Angioarrestin( harp1) SUN Ming2Juan,WANGLiang2Hua,DONG Xiao2Yi,ZONG Ying,GAO Yun,J IAO Bing2Hua 3 ( Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, Shanghai , China) Abstract Angioarrestin is one kind of potential antiangiogenesis factors By DNA recombinant technique to construct E coli BL21 (DE3) expressed plasmid pmal2c 2 2hFD,and hfd cdna fused to the 3πend of the gene encoding the MBP protein The fusion protein was expressed in E coli BL21 (DE3) at 37 after 013 mmolπl IPTG induction for 4 hours The fusion protein of high expression has a molecular weight 66 kd by SDS2PAGE The yield of fusion protein was 20 percent in total proteins measured by SDS2PAGE Supernatant of purified recombinant protein was used as immunogen to prepare the polyclonal antibody The result of ELISA shows that antibody potency is The fusion protein highly expressed in E coli BL21 (DE3) posses well antigen activity And preparation of polyclonal antibody will provide material for further studies of angioarrestin Key words angioarrestin ; MBP ; fusion protein ; E coli expression ; polyclonal antibody (angiopoietins,angs) (1999 ) PCR 21 ( human angiopoietin2 related protein 1,hARP1), [1 ] Angs, ARP1 Tie2,, : , : ,Angs [2 5 ] 2002, Dhanabal [6 ] (No ) 3 Tel : (021) ,E2mail : com cn Received : July 25,2006 ;Accepted : November 1,2006 Supported by Science and Technology Development Foundation of Shanghai (No ) (homology2based gene mining approach) cdna, Kim [7 ] 3 Corresponding author Tel : (021) , E2mail : com cn China Academic Journal Electronic Publishing House All rights reserved

2 2 :Angioarrestin(hARP1) C FD 137 angioarrestin,angioarrestin pmal2c 2 2 FD E coli BL21 (DE3), PCR angioarrestin pmal2c 2 2 FD, pmal2c 2 2 FD E coli BL21 (DE3) [8 ] angioarrestin pmal2c 2 E coli BL21 (DE3) 4 ml LB ( Amp angioarrestin, C FD 100 gπml), cdna 4 ml LB h MBPΠFD A IPTG 013 mmolπml,, h, g angioarrestin [ min, 100 l TE ] SDS2PAGE l, 2, E coli BL21 5 min, SDS2PAGE, 4 %, (DE3) pmal2c 2,pMD182T 12 % 80 V TaKaRa, cdna ; 150 V Invitrogen R Taq DNA 15 min BamH Hind, T4 DNA ml TaKaRa ; HRP, PBS (ph 712) 2 g ( IgG, ) 10 ml (20 mmolπl Tris2HCl,pH HRP IgG 714 ;200 mmolπl NaCl ;1mmolΠL EDTA), MBP NEB (4, rπmin,30 min),sds2page ( ) 215 mgπml, 1 mlπmin BalbΠc,16 18 g 15 ml ( + 10 mmolπl ) PCR [9 12 ] 3 ml 10 GenBank angioarrestin (NM ; GI : ) cdna pmal2c 2 20 l bp angioarrestin C FD, P1 : 5 2GT GGA TCC TTC AAA GAC TGT CAG CA23, P1 5 BamH P2 :5 2GCGAAG CTT TTA CTT GCT PBS TCT GTA ATG GCC23, 5 Hind, g,013 ml, cdna 0105 mlπ ; 10 d PCR, 95 5 min ; 1 1, 95,40 s ;58,40 s ;72,40 s ; 30, min 20 ; nm 5 min SDS2PAGE [15, ], BalbΠc, l g, T 2 angioarrestin pmal2c 2 ( ) 20 % 50 % China Academic Journal Electronic Publishing House All rights reserved

3 % 212 IgG Western, SDS2PAGE pet222b ( + ) 42 kd MBP( ) SDS2PAGE, 66 kd, ( 0145 m MBPΠhFD, 400 ma 45 min) hfd 20 %( Fig11A) MBPΠhFD, ( Fig11B),,DAB ( Fig11A) kd 66 kd 1 ( Fig12A) ; 213 NCI2H460 ( ) pcdna3112mycπhisb ( 2) 2FD NCI2H460 ( SDS2PAGE ), ,, 7 ( Fig11C) :011 molπl PBS 2 3 ;4 % 214 Western, 30 min ; 011 molπl PBS 3 min 3 ;012 % PBST(200 l Triton X2100Π100 ml 011 molπl PBS) 10 min ;011 molπl PBS 3 min 4 ;011 gπml K min ; 4 % 2PBS (ph 712) 5 min ; 011 molπl PBS 3 min 3 ; 3 % Π min ;011 molπl PBS 3 min 3 ; 30 min ; NCI2H460 hfd, ( FD,1 100 pcdna3112mycπhisb ( 2) 2FD NCI2H460 ),50 l, 50 l ( Fig 3), 4 ;011 molπl PBS 3 min 3 ; (,1 25 ) 50 l, 3 1 h ;PBS 3 min 3 ;DAB 10 min ; 2 3,, % ;, 013 mmolπl IPTG MBP hfd pet222b ( + ) hfdπ MBP 21 kd ( Fig 2B) MBPΠFD hfd pmal2c 2 angioarrestin cdna pmal 2 laci ciq 211, IPTG 2 PCR MBP 10 cdna ptrg2xr 560 bp MBP hfd DNA, MBP PCR pmd218t pmd2 GST male 18T2 FD BamH ΠHind, mrna pmal2c 2 GST, pmal2c 2 2 FD, E coli BL21 (DE3) China Academic Journal Electronic Publishing House All rights reserved

4 2 :Angioarrestin(hARP1) C FD 139 Fig 1 Confirmation of MBPΠFD protein by SDS2PAGE analysis (A) Different cell lysate analyzed by SDS2PAGE 1 : E coli BL21 (DE3) ; 2 :Induced pmal2c 2 ΠBL21 ; 3 :Induced pmal2c 2 2hFDΠBL21 ;M:Marker (B) Different pattern cell lysate of BL21(DE3)with pmal2c 2 2hFD plasmid was analyzed by SDS2PAGE 1 : E coli BL21(DE3) ; 2 : Uninduced pmal2c 2 2hFDΠ BL21 ; 3 : Induced pmal2c 2 2hFDΠBL21 ; 4 : Sedimentation of induced pmal2c 2 2hFDΠBL21 ; 5 : Supernatant of induced pmal2c 2 2hFDΠBL21 ; M: Marker (C) Purified protein in different fractions 1 : Fraction 1 eluate from column ;2 : Fraction 2 eluate from column ; 3 : Fraction 3 eluate from column ; 4 : Fraction 4 eluate from column ; 5 : Fraction 5 eluate from column ; 6 : Fraction 6 eluate from column ; 7 : Fraction 7 eluate from column ;M: Molecular weight marker Fig 2 Immunoblotting analysis of tagged hfd recombinant protein (A) The quality of the recombinant protein is checked by rabbit anti2mbp polyclonal antibody 1 : MBPΠFD recombinant protein ;2 : MBP protein (B) The quality of the recombinant protein is checked by mouse anti2mbpπfd polyclonal antibody 1 :pet222b2hfdπbl21 (DE3) ; 2 : E coli BL21 (DE3) ; M:Protein molecular weight marker Fig 3 The FD - ΠFD + NCI2H460 cells checked by immunohistochemistry assay using mouse anti2mbpπfd polyclonal antibody(250 ) (A) Wild type NCI2H460 cell line ; (B) NCI2H460 cells transfected with pcdna3112mycπhisb ( 2) 2FD China Academic Journal Electronic Publishing House All rights reserved

5 140 23, angioarrestin C FD 20 % [17,18 ] 312, [J ] FEBS Lett,1999,443(3) : PBS BalbΠc angioarrestin,,, Western,hFD,, E coli BL (DE3) angioarrestin C FD cdna,, angioarrestin ( References) [ 1 ] Beckner M E Factors promoting tumor angiogenesis [ J ] Cancer Invest,1999,17(8) : [ 2 ] Davis S,Aldrich T H,Jones P F, et al Isolation of angiopoietin21,a ligand for the TIE2 receptor,by secretion2trap expression cloning[j ] Cell,1996,87 (7) : [ 3 ] Hayes A J, Huang W Q, Yu J, et al Expression and function of angiopoietin21 in breast cancer[j ] Br J Cancer,2000,83(9) : [ 4 ] Wurmbach J H, Hammerer P, Sevinc S, et al The expression of angiopoietins and their receptor Tie22 in human prostate carcinoma [J ] Anticancer Res,2000,20(6D) : [ 5 ] Yu Q,Stamenkovic I Angiopoietin22 is implicated in the regulation of tumor angiogenesis[j ] Am J Pathol,2001,158 (2) : [ 6 ] Dhanabal M,LaRochelle W J,Jeffers M, et al Angioarrestin : an antiangiogenic protein with tumor2inhibiting properties [ J ] Cancer Res,2002,62(13) : [ 7 ] Kim I, Kwak H J, Ahn J E, et al Molecular cloning and characterization of a novel angiopoietin family protein,angiopoietin23 [ 8 ] Dhanabal M, Jeffers M, LaRochelle W J, et al Angioarrestin : a unique angiopoietin2related protein with anti2angiogenic properties [J ] Biochem Biophys Res Commun,2005,333 (2) : [ 9 ] Sasse J Detection of proteins in gels[ M]ΠΠAusebel F M, et al ed Current Protocols in Molecular Biology New York : Greene Publishing & Wiley2Interscience 1989 : [10 ] Sambrook J,Fritsch E F,Maniatis T Molecular Cloning :A Laboratory Manual [ M ] 1989 : New York : Cold Spring Harbor Laboratory Press [11 ] Neu H C,Heppel L A The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts[j ] J Biol Chem,1965,240 : [12 ] Smith J A Quantitation of proteins [ M]ΠΠAusebel F M, et al ed Current Protocols in Molecular Biology New York : Greene Publishing & Wiley2Interscience, 1989 : [13 ] Schatz P J, Beckwith J Genetic analysis of protein export in Escherichia coli[j ] Annu Rev Genet,1990,24 : [14 ] Maina C V,Riggs P D, Grandea A G 3rd, et al An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose2binding protein[j ] Gene,1988,74 (2) : [15 ] Hu Y X,Guo J Y,Shen L, et al Get effective polyclonal antisera in one month[j ] Cell Res,2002,12 (2) : [16 ] [ M] : ( Xu Yi2Wei Technique for Immunodetection [ M] Beijing : Science Press),1997, [17 ] Rosenberg A H, Goldman E,Dunn J J, et al Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system[j ] J Bacteriol,1993,175(3) : [18 ] Goldman E, Rosenberg A H, Zubay G, et al Consecutive low2usage leucine codons block translation only when near the 5 end of a message in Escherichia coli[j ] J Mol Biol,1995,245 (5) : China Academic Journal Electronic Publishing House All rights reserved