V o l. 26 N o. 5 2 0 0 5 5 CH EM ICAL JOU RNAL O F CH IN ESE UN IV ERS IT IES 874 879 IL -4 1, 2, 3, 1, 2, 1, 2 (1., 110016; 2., 100039; 3. Beckm an R esearch Institute, C ity of Hope N ationalm edical Center, 1500 D uarte R d. D uarte, CA 91010, U SA ) IL 24 13T, 121R, IL 24 cp IL 4 (13D 121E), PE38KD EL, cp IL 4 (13D 121E) 2PE38KD EL., 30%.,, g IL 24 D audi, IL 24 2, g IL 24. ; 24 ( IL 24); Q 784 A 025120790 (2005) 0520874206, IL 24, IL 24, Burk itt [1, 2 ]., IL 24., IL 24R 3, IL 24R. g IL 24R IL 24R Α IL 22R Χ, T, B. g IL 24R IL 24R Α IL 213R Α1,. g IL 24R IL 24R Α, IL 22R Χ IL 213R Α1, B., IL 24., IL 24 PE, IL 24 IL 24 1% [3 ]. IL 24 N C IL 24,, IL 24. IL 42PE, K reitm an [4 ] IL 24, cp IL 42PE, IL 24 IL 24 10% [4 7 ]. IL 24, cp IL 4. IL 24 IL 24R Α,, IL 24 13 T h r IL 24 IL 24R Α, A sp, IL 24., PCR cp IL 4, cp IL 4 (13D ).,. IL 24,, [ 8 ], 121 A rg Glu, cp IL 4 (13D 121E). cp IL 4 (13D 121E) 2PE38KD EL g IL 24R, IL 2 4 40%,,, cp IL 42PE38KD EL,. : 2004204220. : ( : 20011221203). : (1941 ),,,,. E2m ail: w 2f1@ sina. com
N o. 5 : IL 24 875 1 1. 1 AD 494 (D E3) pet 232a (+ ) N ovagen ; pb luescrip t ( IL 24 cdna ) L i2w eber ; pm D PEg ( PE38KD EL ) ; ECV 2304 ; L 929 ; D audi ; Pyrabest DNA T 4 DNA T akara ; N i2n TA H isb ind R Superflow TM N ovagen ; D EA E2 Sepharo se TM Fast F low Pharm acia. Roche ;. a ( ) : 5 T GT TCGTCCGGA GGTAA CGGCGGTCA C2 AA GT GCGA TA TCA C3 ; b ( ) : 5 CTCA GT T GA GCTCT T GGA GGCA GCAAA GA T GTCT2 GT 3 ; c ( ) : 5 T GCT GCCGCCA T GGA CA CAA CT GA GAA GGAAA CC3 ; d ( ) : 5 CT T GT GA CCGCCGT TA CCTCCGGA CGAA CA CT T T GAA TA T T TCT3 ; e ( ) : 5 TCCCGGCCGCCA T GGA CA CA 3 ; f ( ) : 5 GT TCAAA TCT T T GA T GA TCTCCT G3 ; g ( ) : 5 CA TCAAA GA T T T GAA CA GCCTCA CA 3 ; h ( ) : 5 GCGT T GGGA GCTCT T GGA G2 GCA 3 ; i ( ) : 5 T T TCTCT TCCA T GA TCGTCT T TA GCC3 ; j ( ) : 5 GA TCA T GGAA GA GAAA TA T TCAAA G3. M in i P ro tean R 3 Cell ; Gel Doc2000 (B IO 2RAD ) ; 3K18 (Sigm a ) ; TRAN S2BLO T R SD SEM I2DR Y TRAN SFER CELL (B IO 2RAD ) ; W J23 CO 2 ( H arasaw a) ; 1. 2 IL -4 cp IL 4 (13D 121E) In term ed N J 22100. IL 24, PCR cp IL 4, 3 PCR. 1 PCR a b 1 37 DNA ; 2 PCR c d 38 129 DNA ; PCR, T aq DNA. DNA, b c 3 PCR cp IL 4, 38 1292GGN GG21 37. cp IL 4, PCR, IL 24 13. 1 PCR e f, 2 PCR g h. cp IL 24 (13T h r A sp ), cp IL 4 (13D ). e h 3 PCR, cp IL 4 (13D ), PCR, IL 24 121. 1 PCR e i, 2 PCR j h. e h 3 PCR, cp IL 24 (13T h r A sp, 121A rg Glu), cp IL 4 (13D 121E), N cog Sacg. 1. 3 cp IL 4 (13D 121E) -PE38KD EL cp IL 4 (13D 121E) N cog Sacg, pm D PEg ( PE38KD EL ) Sacg H indg, N cog H indg pet 232a (+ ) T 4 DNA, E. coli DH 5Α. AD 494 (D E3),. 1. 4 cp IL 4 (13D 121E) -PE38KD EL 1% LB OD 600= 016, IPT G 4 h.,, 20 mmo lgl, ph = 719 T ris2hc l, 12 000 rgm in 30 m in, 013 Λm, N i2n TA H isb ind R Superflow TM, 012 ml gm in, [ 5 200 mmo lgl, 015 mo lgl N ac l, 20 mmo lgl T ris2hc l (ph = 719) ],., (Roche) 1 50,, 37 3 h, N H is2t ag
8 76 V o l. 26. D EA E2Sepharo se TM Fast F low (Pharm acia B io tech), Buffer A (20 mmo lgl T ris2hc l, ph = 810), Buffer A Buffer B (20 mmo lgl T ris2hc l, 1 mo lgl N ac l, ph = 810), (T rx tag) cp IL 4 (13D 121E) 2PE38KD EL. 1. 5 W estern blot SD S2PA GE,, IL 24, TM B ( ). 1. 6 IL -4R 125 I IL 24 g IL 24R D audi g IL 24R ECV 2304, cp IL 42PE38KD EL cp IL 4 (13D 121E) 2PE38KD EL. 2 h,, Χ. 1. 7 g IL 24R D audi g IL 24R ECV 2304 96, 1 10 5 g(100 ΛL ), cp IL 4 (13D 121E) 2PE38KD EL, cp IL 42PE38KD EL cp IL 4 (13D 121E), BSA, 5% CO 2, 37 48 h., M T T ( ), 4 h,, DM SO, 570 nm, (% ) = ( A 570g A 570) 100%,. IL 24R L 929. 2 2. 1 cp IL 4 (13D 121E) IL 24 PCR cp IL 4; cp IL 4, 13 (A CT GA T ), cp IL 4 (13D ) ; cp IL 4 (13D ), 121 (A GA GAA ), cp IL 4 (13D 121E)., cp IL 4 (13D 121E) : GA CA CAA CT GA GAA GGAAA CCT TCT GCA GGGCT GCGA CT GT GCTCCGGCA GT TCT A CA GCCA CCA T GA GAA GGA CA CTCGCT GCCT GGGT GCGA CT GCA CA GCA GT TCCA CA GGCA CAA GCA GCT GA TCCGA T TCCT GAAA CGGCTCGA CA GGAA CCTCT GGGGC CT GGCGGGCT T GAA T TCCT GTCCT GT GAA GGAA GCCAA CCA GA GTA CGT T GGAAA A CT TCT T GGAAA GGCTAAA GA CGA TCA T G GAA GA GAAA TA T TCAAA GT GT TC GTCCGGA GGTAA CGGCGGTCA CAA GT GCGA TA TCA CCT TA CA GGA GA TCA TCAAA GA T T T GAA CA GCCTCA CA GA GCA GAA GA CTCT GT GCA CCGA GT T GA CCGTAA CA GA CA TCT T T GCT GCCTCCAA G, GGN GG. IL 24 38 129, IL 24 1 37. 13 A sp, GA T ; 121 Glu, GAA. 2. 2 cp IL 4 (13D 121E) -PE38KD EL ( 1), 65 000 [ pet 232a (+ ) N, 12 000, 65 000 ];, 30%. AD 494gpET 32a (+ ) : (1) N (T rx),, T rx ; (2) IL 24 3, IL 24, T rx 2,,. SD S2PA GE
N o. 5 : IL 24 877, cp IL 4 (13D 121E) 2PE38KD EL 90%,. N 6, N i2n TA H isb ind R Superflow TM, M w 65 000, pet 232a (+ ) 2cp IL 4 (13D 121E) 2PE38KD EL. M w 53 000, cp IL 4 (13D 121E ) 2PE38KD EL. SD S2 PA GE, 95%, 50%. F ig. 1 Expression and pur if ication of cp IL 4 (13D 121E) -PE38KD EL 1. P ro tein m arker; 2. induced AD 494 (D E3) gpet 232a (+ ) ; 3. uninduced AD 494 (D E3) gpet 232a (+ ) 2cp IL 4 (13D 121E ) 2 PE38KD EL ; 4. induced AD 494 (D E3) gpet 232a ( + ) 2cp IL 4 ( 13D 121E ) 2PE38KD EL ; 5. cp IL 4 ( 13D 121E ) 2PE38KD EL fusion p ro tein purified by N i 2+ affinity resin; 6. cp IL 4 ( 13D 121E ) 2PE38KD EL fusion p ro tein purified by anion exchange chrom atography. 2. 3 W estern blot F ig. 2 W estern blot of cp IL 4 (13D 121E) -PE38KD EL 1. U ninduced AD 494 (D E3 ) gpet 232a ( + ) 2cp IL 4 ( 13D 121E) 2PE38KD EL ; 2. induced AD 494 (D E3) g pet 232a ( + ) 2cp IL 4 ( 13D 121E ) 2PE38KD EL ; 3. cp IL 4 ( 13D 121E ) 2PE38KD EL fusion p ro tein purified by N i 2+ affinity resin; 4. cp IL 4 (13D 121E) 2 PE38KD EL fusion p ro tein purified by anion exchange ch rom atography; 5. SeeB lue R P re2stained P ro tein M arker. 2, AD 494 (D E3) gpet 232a (+ ) 2cp IL 4 (13D 121E) 2PE38KD EL cp IL 4 (13D 121E) 2PE38KD EL 65 000, cp IL 4 (13D 121E) 2PE38KD EL ; cp IL 4 (13D 121E) 2PE38KD EL 53 000, cp IL 4 (13D 121E) 2PE38KD EL ; AD 494 (D E3) g pet 232a (+ ). 2. 4 IL -4R, D audi, cp IL 4 (13D 121E ) 2PE38KD EL IL 24 cp IL 42PE38KD EL 4. ECV 2304, cp IL 4 (13D 121E) 2PE38KD EL IL 24 cp IL 42PE38KD EL 1g3. 1. M o lecule Table 1 Binding of IL 4-tox in s to IL -4R-bear ing cells D audi cell EC50g(nmo l L - 1 ) A ffinity of IL 24 (% ) ECV 2304 cell EC 3 50 g(nmo l L - 1 ) A ffinity of IL 24 (% ) IL 24 01068 10010 01037 10010 cp IL 42PE38KD EL 01540 1216 01320 1116 cp IL 4 (13D 121E) 2PE38KD EL 01150 4513 01830 415 EC50: the imm uno toxin concentration necessary fo r 50% disp lacem ent of 125 I2labeled IL 24 from the cells. 2. 5,, cp IL 4 (13D 121E) 2PE38KD EL, D audi ; cp IL 4 (13D 121E) 2PE38KD EL cp IL 42PE38KD EL 3, BSA cp IL 4 (13D 121E) [ 3 (A ) ]. cp IL 4 (13D 121E) 2PE38KD EL ECV 2304,, ; cp IL 4 (13D 121E) 2PE38KD EL IL 24R L 929 [ 3 (B ) ]. IL 24 4 Α, N C A, B, C, D. IL 24
8 78 V o l. 26 F ig. 3 The cytotox ic ity of the IL -4 immunotox in (A ) T he cyto toxicity of the IL 24 imm uno toxins to D audi; (B) the cyto toxicity of cp IL 4 (13D 121E) 2PE38KD EL to D audi, ECV 2304 and L 929., A, C IL 24R Α, D IL 24R ( IL 22R Χ IL 213R Α1), [9, 10 ]. IL 24 N I5, E9, T 13 C R 121, Y124, S125., IL 24 C PE, IL 24., K reitm an [4 ] cp IL 24, IL 24 N C GGN GG, (37 38 ) N C. Sw iss PDB V iew IL 24 IL 24R Α, IL 24 IL 24R Α 0. 3 nm : IL 24 T 6, E9, Q 78, R 81, R 85, R 88, IL 24R Α Y13, D 67, S70, D 72, D 125, Y183., IL 24 IL 24R Α,, : IL 24 R 88 IL 24R Α D 72, IL 24 E9 IL 24R Α Y183 [11 ], 013 nm. IL 24, IL 24 A C IL 24R Α, K off IL 24 Glu29 A rg288, 5 Ile25, T h r213, A rg253, A sn289, T rp 291. K on C 5 (L ys277, A rg281, L ys284, A rg2 85, A rg288) 2 (Glu29 T h r213) [12 ]. IL 24 IL 24R Α, 013 014 nm : IL 24 I5, K12, T 13, R 53, Y56, D 87, N 89, W 91., 13 T h r. IL 24 IL 24R Α ( 4), IL 2 4 13 IL 24R Α 127 T yr, 014 nm, 13 A sp, A sp, IL 24, T yr, IL 24 IL 24R Α., 13 A sp, A sp IL 24R Α. Srivannaboon [13 ], IL 24 13 A sp, IL 24 IL 24R 18. W ang [12 ], IL 24 132T h r A la, 3 ; 132T h r A sp, 3. F ig. 4 In teraction s of IL -4 (13D ) and IL -4R Α (A ) Interactions of D 13 w ith adjacent am ino acids from IL 24R Α; (B) Interactions of E9 and D 13 on A 2helix of IL 24 w ith adjacent am ino acids from IL 24R Α.
N o. 5 : IL 24 879 cp IL 24 IL 24, cp IL 24 13 T h r A sp, cp IL 24 IL 24R Α, IL 24. IL 24, IL 24 D 121 A rg Glu, IL 24 T B, [8 ]. cp IL 24,,,,,., cp IL 24 13 121 cp IL 24 (13D 121E) 2PE38KD EL IL 24R cp IL 242PE38KD EL 3,,. [ 1 ] Kreitm an R. J., Puri R. K., Pastan İ. Cancer Res. [J ], 1995, 55 (15) : 3357 3363 [ 2 ] D ebinsk iw., Puri R. K., Pastan İ. Inṫ J. Cancer[J ], 1994, 58 (5) : 744 748 [ 3 ] D ebinsk iw., Puri R. K., Kreitm an R. J. et a l.. J. B io l. Chem. [J ], 1993, 268 (19) : 14065 14070 [ 4 ] Kreitm an R. J., Puri R. K., Pastan İ. P roc. N atl. A cad. Sci., U SA [J ], 1994, 91 (15) : 6889 6893 [ 5 ] Puri R. K., Hoon D. S., L eland P. et a l.. Cancer Res. [J ], 1996, 56 (24) : 5631 5637 [ 6 ] Puri R. K., M eh ro tra P. T., L eland P. et a l.. J. Imm uno l. [J ], 1994, 152 (7) : 3693 3700 [ 7 ] H usain S. R., Behari N., Kreitm an R. J. et a l.. Cancer Res. [J ], 1998, 58 (16) : 3649 3653 [ 8 ] Shanafelt A. B., Fo rte C. P., Kasper J. J. et a l.. P roc. N atl. A cad. Sci., U SA [J ], 1998, 95 (16) : 9454 9458 [ 9 ] W alter M. R., Kood W. J., Zhao B. G. et a l.. J. B io l. Chem. [J ], 1992, 267 (28) : 20371 20376 [ 10 ] H age T., Sebald W., Reinem er P.. Cell[J ], 1999, 97 (2) : 271 281 [ 11 ] Zhang J. L., Sim eonow a İ, W ang Y. et a l.. J. M o l. B io l. [J ], 2002, 315 (3) : 399 407 [ 12 ] W ang Y. H., Shen B. J., Sebald W.. P roc. N atl. A cad. Sci., U SA [J ], 1997, 94 (5) : 1657 1662 [ 13 ] Srivannaboon K., Shanafelt A. B., Todisco E. et a l.. B lood[j ], 2001, 97 (3) : 752 758 Im provem en t of Selectiv ity and Cytotox ic ity of IL -4 Imm unotox in on L ym phoma Through Site-d irected M utagenesis CU I J ing2x ia 1, 2, J I J ian2fei 3 1,, L U g A n2guo 2 1, 23, W U W en2fang (1. S heny ang Institu te of A pp lied E cology, Ch inese A cad em y of S ciences, S heny ang 110016, Ch ina; 2. G rad uate S chool of the Ch inese A cad em y of S ciences, B eij ing 100039, Ch ina; 3. B eckm an R esearch Institu te, C ity of H op e N ational M ed ical Center 1500 D uarte R d. D uarte, CA 91010, U SA ) Abstract A w ide variety of hum an cancer cells such as gliom a and lymphom a exp ress in terleuk in24 recep to rs ( IL 24R ), therefo re, it m ay be a good op tion to treat IL 24R 2bearing tumo r w ith IL 242 con tain ing imm uno tox in s. By softw are analysis, tw o impo rtan t am ino acids 13T and 121R of IL 24 w ere cho sen fo r site2directed m u tation. In terleuk in24 m u tein cp IL 24 (13D 121E) w as ob tained th rough overlapp ing PCR and the ch im eric imm uno tox in w as con structed by fu sion of the gene encoding cp IL 2 4 (13D 121E ) to a gene encoding a modified P seudomonas exo tox in A (PE38KD EL ). T he ch im eric imm uno tox in w as exp ressed in E. coli w ith the yield of abou t 30% of the to tal bacterial p ro tein. A fter being h igh ly purified by affin ity ch rom atography and an ion exchange ch rom atography, the ch im eric p ro tein w as tested fo r its cyto tox icity. T he data show that cp IL 24 ( 13D 121E ) 2PE38KD EL had imp roved cyto tox icity to lymphom a cells D audi exp ressing class g IL 24R in comparison w ith o ther IL 2 42con tain ing imm uno tox in and had a low er cyto tox icity to endo thelial cells exp ressing class g IL 24R. Keywords Imm uno tox in; In terleuk in24 ( IL 24R ) ; P seudomonas exo tox in A (Ed. : H, J, Z)