IL -4 . IL 24, cp IL 42PE38KD EL,.
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1 V o l. 26 N o CH EM ICAL JOU RNAL O F CH IN ESE UN IV ERS IT IES IL -4 1, 2, 3, 1, 2, 1, 2 (1., ; 2., ; 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 (2005) , 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 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 %,,, cp IL 42PE38KD EL,. : : ( : ). : (1941 ),,,,. E2m ail: w 2f1@ sina. com
2 N o. 5 : IL 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 IL 24, PCR cp IL 4, 3 PCR. 1 PCR a b 1 37 DNA ; 2 PCR c d DNA ; PCR, T aq DNA. DNA, b c 3 PCR cp IL 4, GGN GG cp IL 4, PCR, IL 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 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 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), cp IL 4 (13D 121E) -PE38KD EL 1% LB OD 600= 016, IPT G 4 h.,, 20 mmo lgl, ph = 719 T ris2hc l, rgm in 30 m in, 013 Λm, N i2n TA H isb ind R Superflow TM, 012 ml gm in, [ 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
3 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 W estern blot SD S2PA GE,, IL 24, TM B ( ) 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 g IL 24R D audi g IL 24R ECV , g(100 ΛL ), cp IL 4 (13D 121E) 2PE38KD EL, cp IL 42PE38KD EL cp IL 4 (13D 121E), BSA, 5% CO 2, h., M T T ( ), 4 h,, DM SO, 570 nm, (% ) = ( A 570g A 570) 100%,. IL 24R L 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 , IL A sp, GA T ; 121 Glu, GAA cp IL 4 (13D 121E) -PE38KD EL ( 1), [ pet 232a (+ ) N, , ];, 30%. AD 494gpET 32a (+ ) : (1) N (T rx),, T rx ; (2) IL 24 3, IL 24, T rx 2,,. SD S2PA GE
4 N o. 5 : IL , cp IL 4 (13D 121E) 2PE38KD EL 90%,. N 6, N i2n TA H isb ind R Superflow TM, M w , pet 232a (+ ) 2cp IL 4 (13D 121E) 2PE38KD EL. M w , 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 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 , cp IL 4 (13D 121E) 2PE38KD EL ; cp IL 4 (13D 121E) 2PE38KD EL , cp IL 4 (13D 121E) 2PE38KD EL ; AD 494 (D E3) g pet 232a (+ ) 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 cp IL 42PE38KD EL cp IL 4 (13D 121E) 2PE38KD EL 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
5 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, 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 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 A sp, IL 24 IL 24R 18. W ang [12 ], IL T 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.
6 N o. 5 : IL cp IL 24 IL 24, cp IL 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 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) : [ 2 ] D ebinsk iw., Puri R. K., Pastan İ. Inṫ J. Cancer[J ], 1994, 58 (5) : [ 3 ] D ebinsk iw., Puri R. K., Kreitm an R. J. et a l.. J. B io l. Chem. [J ], 1993, 268 (19) : [ 4 ] Kreitm an R. J., Puri R. K., Pastan İ. P roc. N atl. A cad. Sci., U SA [J ], 1994, 91 (15) : [ 5 ] Puri R. K., Hoon D. S., L eland P. et a l.. Cancer Res. [J ], 1996, 56 (24) : [ 6 ] Puri R. K., M eh ro tra P. T., L eland P. et a l.. J. Imm uno l. [J ], 1994, 152 (7) : [ 7 ] H usain S. R., Behari N., Kreitm an R. J. et a l.. Cancer Res. [J ], 1998, 58 (16) : [ 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) : [ 9 ] W alter M. R., Kood W. J., Zhao B. G. et a l.. J. B io l. Chem. [J ], 1992, 267 (28) : [ 10 ] H age T., Sebald W., Reinem er P.. Cell[J ], 1999, 97 (2) : [ 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) : [ 12 ] W ang Y. H., Shen B. J., Sebald W.. P roc. N atl. A cad. Sci., U SA [J ], 1997, 94 (5) : [ 13 ] Srivannaboon K., Shanafelt A. B., Todisco E. et a l.. B lood[j ], 2001, 97 (3) : 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 , Ch ina; 2. G rad uate S chool of the Ch inese A cad em y of S ciences, B eij ing , 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)
Supplemental file 3. All 306 mapped IDs collected by IPA program. Supplemental file 6. The functions and main focused genes in each network.
LIST OF SUPPLEMENTAL FILES Supplemental file 1. Primer sets used for qrt-pcr. Supplemental file 2. All 1305 differentially expressed genes. Supplemental file 3. All 306 mapped IDs collected by IPA program.
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