T. Kaw akam i. B ru ton. , Sf9

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1 14 3 V o l114,n o Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy Jun 1998 Bruton T Kaw akam i (L a Jo lla Institute fo r A llergy and Imm uno logy, San D iego, CA, U SA ) ( ;, ) B ru ton (B ru ton tyro sine k inase,b tk), B tk H 902 DNA Sf9,, H 902, B tk B tk, B tk : B ru ton,, Sf9 Expression of Bruton Tyrosine K ina se in Sf9 In sect Cells H an W ei Yao L i2bo To sh iak i Kaw akam i (L a J olla Institu te f or A llergy and Imm unology, S an D ieg o, CA, U SA ) (B iotechnique Center, B iochem istry D ep artm ent, the 4th M ilitary M ed ical U niversity, X iπan ) Abstract T he p resen t study w as undertaken to exp ress m ou se B ru ton tyro sine k inase (B tk) by bacu loviru s exp ression vecto r system in Sf9 in sect cells In o rder to detect and purify B tk p ro tein, the H 902 sequence w as in serted in up stream sequence of the in itiation codon of B tk gene T hen H 902 B tk gene w as cloned in to bacu loviru s tran sfer vecto r (pvl 1393) Sf9 cells w ere co tran sfected w ith linear au tographa califo rn ica nuclear po lyhedro sis viru s (A cn PV ) DNA and pvlb tk tran sfer vecto r u sing cation ic lipo som es fo r 5 days A fter 3 round am p lifi2 cation of recom b inan t bacu loviru s, the cell lysates w ere analyzed by sodium dodecyl su lfate po lyacrylam ide gel electropho resis and b lo tted on to po lyvinylidene difluo ride m em b raneṡ T he m em b ranes w ere incubated w ith p rim ary and secondary an tibodies and fo llow ed by de2 tection w ith an enhanced chem ilum inescence k iṫ T he resu lt show ed that a h igh exp ression level of B tk p ro tein in Sf9 cells infected by the recom b inan t bacu loviru s w as detected by im 2 m unob lo tting w ith H 902 an tibody T he p ro tein au topho spho rylation assay also show ed that B tk p ro tein exp ressed by in sect cells had a stab le au topho spho rylation level Key words: B ru ton tyro sine k inase,bacu loviru s exp ression vecto r system, Sf9 cell B ru ton (B tk) B tk 77 kd, X B tk [ 1, 2 ] [ 3, 4, X B tk ], B tk, B tk : , :

2 [ 5, 6 B tk ] : 1) IgE (FcΕR I), B tk ; 2)B tk C, C B tk, B tk, B tk B tk Sf9 ( ) pm E B tk ( B tk ) pvl 1393 ( ), N ew England B iol ab s, Invitrogen, TNM 2FH PharM igen, Imm ob ilon2p ( 0145 Λm ) M ilipo re,w estern b lo tting Am ersham, [ Χ2 32 P ]2dA T P ICN, B ior ad, H 902 ( 1g 500), HR P2 IgG ( 1g5000) F isher 112 pm EH902 Btk H 902 : B tk, B tk ( 1 )N co lg H bp, N cog (F ig 1) Am erican Cu stom er F ig 1 N ucleo tide sequence of H 902 [ : H 902 ] pm EB tk DNA N cog H 902,, DNA, 113 Btk ( H902 ) (pvl 1393) N o tg Speg pm EH 902 B tk DNA, 018% B tk ( H 902), N o tg Xbag pvl 1393, [ Sf9 Invitrogen ] 115 Stefan [ 9 ], 116 Btk Sf9 Sf9 ( ) 115 m l, (1% N P240 1 mm o lgl N a2o 2vana2 date, 1 mm o lgl ED TAg2N a, 10 m ggm l leupep tin, 011 m o lgl PM SF, 5 m ggm l ap ro tin in) 600 Λl, 20 s, 10 m in, 4 ( rgm in) 10 m in, ( B io2r ad ) 1 Λg, 8% SD S2PA GE Imm ob ilon2p, 2% gelatin 37 1 h, PBS2Tw een H 902, 1 h, 4, 30 m in, ECL, Am ersham πs,, 270

3 , X, 117 Btk B tk Sf9 ( ) (PVL 1393) Sf9 ( ) 115 m l, ( ), m l ( 60 Λg), 1 Λg H 902, 4 2 h, 30 Λl 10% pan so rb in, 4 30 m in rgm in 2 m in, PT yr (50 mm o lgl H EPES ph 714, 10% T riton2x mm o lgl N ac l, 100 mm o lgl N af, 2 mm o lgl N a2o 2vanadate), PKS (10 mm o lgl T risghc l ph 712, 10 mm o lgl M nc l2), 20 Λl A (50 mm o lgl T risghc l ph 712, 10 mm o lgl M nc l2, 011% N P240, 20 Λl B (50 mm o lg L T risghc l ph 712, 10 mm o lgl M nc l2, 011% N P Λm o lgl A T P, 10 ΛC i [Χ2 32 P ]2A T P), m in,, 3 m in SD S2PA GE ( 8% ), Imm ob ilon2p pm E H902 Btk, pm E H 902 B tk, 5 F ig 2, X H 902 B tk, H 902, pm E H 902 B tk 212 pvl H902 Btk, pvl H 902 B tk DNA DNA, N o tg Xbag (B tk ),, 214 kb DNA F ig 3, 5 F ig 2 N ucleo tide sequence of pm E H 902 B tk Initiation sequence of B tk is underlined, 3 H indg 3, 3 H 902B tk (pvl 1393) 213 Btk Sf9 Sf9 B tk, (F ig 4), Sf9, B tk,, B tk, B tk,, B tk, B tk, [ 9, B tk ],,,,,, B tk H 902 B tk H 902, B tk B tk 271

4 F ig 3 Gel electropho retic analysis of recom binant pvl 1393 H 903 B tk 11ΚDNA digested by H ind g ; 2 61D ifferent recom bi2 nant pvl 1393 H 902 B tk p lasm id DNA digested by N o t g + Xbag F ig 4 Exp ression of B tk p ro tein in Sf9 cells 11Sf9 cell lysate from co transfection; 21Sf9 cell lysate from first round amp lification of recom binant baculovirus; 31Sf9 cell lysate from second round amp lfication of recom binant baculovirus; 41Sf9 cell lysate from third round amp lfication of recom binant baculovirus; 51Sf9 cell lysate from fourth round amp lification of recom binant baculovirus 214 Btk B tk, Sf9 B tk B tk, [ Χ2 32 P ]2A T P B tk, B tk, [Χ2 32 P ]2A T P, Sf9 B tk (F ig 5),, pvl 1393 Sf9 B tk, B tk Sf9, 77 kd,, B tk, Sf9 B tk Sf9 B tk B tk, B tk,, F ig 5 A utopho soho rylation activity of B tk p ro tein in, ( Sf9 cells 11N egative contro l: Sf9 cell lysate from th ird round am 2 ) p lification of pvl 1393 vecto r (after transfection) ; 21Sf9 [ 10 ], 272 cell lysate from third round amp lification of recom bina2 tion B tk baculovirus

5 References 1 V etrie D, V o rechovsky I, Sideras P, Ho llend J,Davies A, F linter F, H amm arstrom L, Kinnon C, L evinsky R, Bobrow M, Sm ith C I E, Bentley D R T he gene invo lved in X2linked a gamm a globulinem ia is a m em ber of the src fam ily of p ro tein2 tyro sine k inase N atu re, 1993, 361: T sukada S, Saffran D C, Raw lings D J, Paro lini O, A llen R C, Klisak I, Sparkes R S, Kubagaw a H,M ohandas, T, Q uan S, Belmont J W, Cooper M D, Conley M E,W itte O N D eficient exp ression of a B cell cytop lasm ic tyro sine k inase in hu2 m an X2linked agamm aglobulinem ia Cell, 1993, 72: T hom as J D, Sideras P, Sm ith C I E, V o rechovsky I, Chapm an V, Paul W E S cience, 1993, 261: Raw lings D J, Saffran D C, T sukada S, L argaespada D A, Grim aldi J C, Cohen L, M oh r R N, Bazan J F, How ard M, Copeland N C, Jenk ins N A,W itte O N M utation of unique region of B rutons tyro sine k inase in imm unodeficient X ID m ice S cience, 1993, 261: Yao L B, Kaw akam i Y, Kaw akam i T T he p lecstrin homo logy dom ain of B ruton tyro sine k inase interacts w ith p ro tein k i2 nase C P roc N atl A cad S ci U SA, 1994, 91: Kaw akam i Y, Yao L B,M iura T, T sukada S,W itte O N, Kaw akam i T T yro sine pho spho rylationand activation of B ruton tyro sine Kinase upon Fc (R I cro ss2link ing M ole Cell B iol, 1994, 14: Sam brook J, F ritsch E F andm aniatis T M olecu lar C loning: a laboratory m anual, 2nd ed N ew Yo rk: Co ld Sp ring H arbo r L abo rato ry P ress, Co ld Sp ring H arbo r, 1989: Inv itrog en M axb ac 210 T ransf ection and E xp ression M anual Catalog no K K835202, K875202, K Gruenw ald S, H eitz J B acu lov irus E xp ression V ector S y stem : P roced u res and M ethod s M anual, 2nd ed PharM ingen, 1993: Javis D L, Summ er M D Glyco sylation and secretion of hum an tissue p I asm iogen activato r in recom binant baculovirus2 infected cells M ol Cell B iol, 1989, 9: