Expression of Functiona l D oma in g of Ca lpa sta tin in E. coli and Prepara tion of An tiserum Aga in st Pur if ied GST-Ca lpa sta tin

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1 ISSN CN gQ 16 1 Ch in. J. B iochem. M o l. B io l. 2000, V o l. 16, N o. 1, g 3 1) 2) 2) 1) ( 1), 2), ) PCR (calpastatin) cdna g (404 bp ), pgex2kg pgex2calp 4 IPT G 45 kd GST 2Galp 4 2Sepharo se 4B GST 2Calp 4, GST 2Calp 4 8, 1 64W estern2b lo t 140 kd ( calpastatin),,, GST,, Q 503,Q 78 Expression of Functiona l D oma in g of Ca lpa sta tin in E. coli and Prepara tion of An tiserum Aga in st Pur if ied GST-Ca lpa sta tin DU M in 1), ZHU M ei2jun 2), ZHAN G M an2fu 2),NAN Q ing2x ian 1) ( 1) Colleg e of F ood S cience, 2) Colleg e of B iolog ical S ciences, Ch ina A g ricu ltu ral U niversity, B eij ing ) Abstract L o ts of researches have suggested that calpain system p lays an im po rtan t ro le in nu2 m erou s cell function s, bu t its m echan ism is still no t clear now. In o rder to fu rther research, the con servative dom ain g,w ith inh ib ito ry activity,w as am p lified by PCR and cloned in to exp ression vecto r pgex2kg and tran sfo rm ed E. coli. A fter 3 h induced by IPT G, the exp ression of recom b i2 nan t 45 kd GST 2Calp 4 fu sion p ro tein w as confirm ed by SD S2PA GE. GST 2Calp 4 w as purified from lysates w ith glu tath ione Sepharo se 4B co lum n. T he purified GST 2Calp 4 w as m ixed w ith F reund s com p lete o r incom p lete adjuvan t, and then u sed to imm un ize rabb itṡ E igh t w eek s later, the fiter of an tiserum w as T he IgG fo rm w as then purified by fractionation w ith (N H 4) 2SO 4. W hen ex tracted p ro tein from m u scle cells w as app lied fo r W estern2b lo t analysis, there is on ly one band around 140 kd w as detected,w h ich is the sam e as the m o lecu lar w eigh t of com p lete calpastatin. Key words Calpastatin, P rokaryo tic exp ression, GST fu sion p ro tein, A ffin ity ch rom atography, A n tiserum [ 2 ] ;,, Ca 2+, (calpain) (calpastatin), 3 ( ) (E254721) 3 3,, ,,,, T el: (010) , Fax: (010) [ 1 ], calpastatin E2m ail: D um in@ho tm ail. com : , :

2 24 16 [ 3 calpastatin ], SD S2PA GE, m GST 2Calp 4 calpain, Pharm acia, : IPT G [ 4 calpastatin ],,,, 2Sepharo se 4B, 10 mm o lgl,, GST 2Calp 4, GST 2Calp 4 [ 5 ], GST 2Calp 4 ; IgG calpastatin 11215, Calpastatin, L, 1 4, [ 6 ] calpastatin calpastatin g,, GST 2Calp 4 calpastatin calpastatin, GST 2Calp 4, calpastatin, calpastatin cdna Em o ri ; pgex2kg 11112, kg calpastatin, 1: 5 2A CGGA TCCAAA T T TCT GCT 23 ; 2: 5 2GCT GAA T TCT GT GAA GTCTCT G23 1, 1, 2 5 B am H g E cor g Calp 4 GST IPT G PCR pgex2calp 4 [7 ] pgex2calp 4 E. coli JM mm o lgl IPT G, 37 3 h, 1215% F ig. 1 Construction of recom binant exp ression vecto r pgex2calp4 (a) The m ap of constructed pgex2calp4 exp ression vecto r; (b) Restriction enzym e analysis of pgex2calp4 exp ression vecto ṙ 1: B am H g ; 2: B am H g + E cor g ; 3: E cor g ; 4: Κ2DNA H ind g M arker

3 1 : g 25 (L ong issim us d orsi), PBS, ph 7. 3, Ph ilip s HR m in, 4, g, 4 20 m in, SD S2 PA GE W estern Tow b in [ 8 ], (GST 2Calp 4 ) 1 500, IgG (Sigm a ), , NBT gbc IP pgex-ca lp4 PCR calpastatin (404 bp ), B am H g E cor g, pgex2kg, pgex2calp 4 (F ig. 1a) pgex2calp 4 B am H g E cor g, 510 kb 404 bp (F ig. 1b), calpastatin 212 pgex-ca lp4 E. coli 213 pgex-ca lp4 F ig. 2, IPT G, (F ig. 3) calpastatin cd 2 pgex2kg JM kd NA [ 9 ],, GST, pgex2calp 4 JM 109 IPT G, 45 kd, 26 kd, calpastatin, GST F ig. 2 IPT G induced exp ression of GST 2Calp4 fusion p ro2 tein in E. coli 1: P ro tein m arker; 2-4: 45 kd GST 2Calp4 exp ression in2 duced by IPTG; 5: Contro l; 6: 26 kd GST exp ression in2 duced by IPTG F ig. 3 Sequence of recom binant exp ression p lasm id pgex2calp4 3 : Sequence in shadow is sequences of pgex2kg 214 GST-Ca lp4 Calp 4 F ig. 4, GST 2Calp 4 215,, 2Sepharo se 4B GST W estern, GST 2 Calp 4, GST 2 Calp 4 140kD

4 26 16 F ig. 4 Purification of GST 2Calp4 fusion p ro tein 1: To tal p ro teins of induced E. coli transfo rm ed w ith pgex2 Calp4; 2: P ro tein sonicated supernatant of induced E. coli trans2 fo rm ed w ith pgex2calp4; 3: P ro tein sonicated pellet of induced E. coli transfo rm ed w ith pgex2calp4; 4: GST 2Calp4; 5: P ro tein m arker (F ig. 5), GST 2Calp 4 1, F ig. 5 Calpastatin identified in m uscle supernatant by W estern2blo tting (a) SD S2PA GE (7. 5% gel) of m uscle cell extraction; (b) Imm unodetection of calpastatin in m uscle cell extraction w ith GST 2Calp4 antiserum; P ro tein m arker are indicated on the left 3,,,, 1, calpastatin,, Calpastatin [ 10 ], pgex2kg GST 2Calp 4,, GST, calpas2 tatin g [ 6 ], GST 2Calp 4 IPT G GST 2Calp 4, GST 2Calp 4, GST 2Calp 4 ( ) 45 kd, (40 kd ), calpastatin [ 10 ] W estern, GST 2 Calp kd, cal2 [ 11, 12 pastatin ] calpas2 tatin calpastatin (References) 1 Sh ields D C, Banik N L. U p regulation of calpain activity and ex2 p ression in experim ental allergic encephalom yelitis: A putative ro le fo r calpain in dem yelination. B rain R es, 1998, 794: L i J, N ixon R, M esser A, Berm an S, Bursztajn S. A ltered gene exp ression fo r calpaingcalpastatin system in mo to r neuron de2 generation (M ND ) m utant mouse brain and sp inal co rd. B rain R es M ol B rain R es (M BR ), 1998, 53: Zalew ska T, Zablocka B, Saido T C, Zajac H, Dom anska2janik K. D ual response of calpain to rat brain po stdecsap itative is2 chem ia. M ol Chem N eu rop athol, 1998, 33: H uang J, Fo rskerg N E. Ro le of calpain in skeletal2m uscle p ro2 tein degradation. P roc N atl A cad S ci U SA, 1998, 95:

5 1 : g 27 5 Kinbara K, Ish iura S, Tom ioka S, So rim ach i H, Jeong S Y, A 2 m ano S, Kaw asak i H, Ko lm erer B, Kim ura A, L abeit S, Suzuk i K. Purification of native p94, a m uscle specific calpain, and characterization of its auto lysis. B iochem J, 1998, 335: ,, C roall D E, D em artino G W. Calcium 2activated neutral p ro tease (D u M in. N an Q ing2xian, Zhu M ei2jun. T he structure and activity regulation of calpain. P rog B iochem B io2 p hy s), 1998, 25: Sam brook J, F ritsch E F, M aniatis T. M olecu lar C loning: A L aboratory M anual, 2nd ed. N ew Yo rk: Co ld Sp ring H arbo r L abo rato ry P ress, Tow bin H, Stahelin T, Go rdon J. E lectropho retic transfer of p ro tein from po lyacrylam ide gel to nitrocellulo se sheet, p roce2 dure and som e app lications. P roc N atl A cad S ci U SA, 1979, 76: Ish ida S, Emo ri Y, Suzuk i K. Rat calpastatin has diverged p ri2 m ary sequence from o ther m amm alian calpastatins but retains functionally impo rtant sequences. B ioch im B iop hy s A cta, 1991, 1088: ( calpain ) system: structure, function, and regulation. P hy siol R ev iew s, 1991, 71: Cong M, T homp son V F, Go ll D E, A ntin P B. T he bovine cal2 pastatin gene p romo to r and a new N 2term inal region of the p ro2 tein are targets fo r cam P2dependent p ro tein k inase activity. J B iol Chem, 1998, 273: Rom an L, H ruska U S. A n imp roved purification p ro toco l fo r heart and skeletal m uscle calpastatin reveals two isofo rm s re2 sulting from alternative sp licing. A rch B iochem B iop hy s, 1998, 356: 19 24