Fusion expression of ra inbo w trout ( O ncor hyncus mykiss) insulin2like growth factors and and their mitogenic activities in E.
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- Ἄμπελιος Αλεβίζος
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1 49 (2) : , 2003 A cta Zoologica S inica 3 33 (, ) Fusion expression of ra inbo w trout ( O ncor hyncus mykiss) insulin2like growth factors and and their mitogenic activities in E. coli 3 ZHAN G Wei2Min ZHAN G Li2Hong ( School of L if e Sciences, Zhongshan U niversity, Guangz hou , Chi na) Abstract The preparation of recombinant rainbow trout insulin2like growth factors ( IGF2 ) and ( IGF2 ) using the His6 fusion polypeptide technique and the mitogenic activities of these compounds were investigated. Rainbow trout IGF2 and IGF2 B2C2A2D domain cdna were subcloned into expression vector p ET215b (Novagen, USA) and ex2 pressed in host E. coli BL21 (DE3) as fusion polypeptides containing a stretch of 6 histidines at the N2terminus (referred as His6 fusion polypeptide). The addition of IPTG (014 mmol/ L) induced the expression of polypeptides with a molecular weight of about 1010 kda. The expressed proteins were ammonium sulfate fractionally precipitated and further purified using a Ni 2 + 2His Bind Resin (Novagen, USA) affinity column. The reduced SDS2PA GE showed that IGF2 was of 80 % purity and IGF2 was of 90 % purity. The rainbow trout IGF2 His6 fusion polypeptide showed dose2dependent mitogenic effects on BALB/ N IH3 T3 cells in the serum free medium culture, and rainbow trout IGF2 His6 fusion polypeptide also showed this kind of activity, but with lower potency. Taken together, these results indicated that the rainbow trout IGF2 and His6 fusion polypeptides produced in E. coli were biologically active, with IGF2 His6 fu2 sion polypeptide of higher potency [ Acta Zoologica Sinica 49 (2) : , 2003 ]. Key words Rainbow trout ( Oncorhynchus mykiss), IGF2, IGF2, His6 fusion polypeptide, Mitogenic activity His6 ( Insulin2like growt h IGF, factor, IGF2 ) ( Insulin2like growt h factor, IGF2 ) B2C2A2D, : IGF2 IGF ( Duan, IGF2 1997) IGF ( Humbel, 1990 ; LeRoith et al., 2001), IGF, IGF, IGF2 IGF, IGF mrna, IGF IGF2,, IGF (Duan, 1998), , (No ) ( ) [ This study was funded by Natural Science Foundation of Guangdong Province (No ) and State Programme for Skeleton Teachers in Higher Institution ] 33 (Corresponding author) E2mail : 36,,, : ν 2003 Acta Zoologica Sinica
2 2 : 267 IGF2 IGF2 IGF GCA23 ( IGF2 ) ; GGC A GG23 ( IGF2 ) IGF, IGF ( Fine et al., 1997 ; Moriyama et al., 1993 ; Upton et al., 1997 ;, 2001), al., 1992) Nde B am H,, pbluescript SK, p ET215b GST IGF, DNA (Chen et al., 2000 ;, 2001 ;, 2002), GST IGF p ETIGF2 p ETIGF2 ( Chen et al., 2000), His6, 6 N - C - 6, BL21 ( DE3 ) p ET215b, p ETIGF2, p ETIGF2 BL21 (Lindner (DE3), p ET215b 5 ml et al., 1997 ; Reeth et al., 1998 ;, LB (75 g Ampicilin/ ml), 37 OD ) IGF2 IGF2 019, 4, His6, 100 ml LB ( Ampicilin 75 g/ ml) IGF2 IGF2 His6, 37 OD , IPTG, BALB/ 014 mmol/ L N IH3 T Sigma, 20 mmol/ L Tris2HCl (p H 719) / 2 mmol/ L ED TA/, New England Biolab Gibco2 BRL F12/ DMEM Gibco2BRL, Sigma, IGF2 20 % 40 % 60 % 80 % 95 % IGF2 Promega p ET2 40 % 60 % 15b His Bind Novagen 1 Binding buffer (5 mmol/ L Imida2 112 Vent DNA, 719), 1125 ml Ni 2 + 2His Bind cdna ( Connecticut T. ( Novagen) T. Chen ), PCR IGF2 IGF2 B2C2A2D cdna PCR Binding buffer, 1 Washing buffer ( 60 mmol/ L IGF B 5 D 5 Imidazole, 015 mol/ L NaCl, 20 mmol/ L Tris2HCl, : 5 2GA G A GA CA T A TG GGG CCC GA G ACC CTG23 ( IGF2 ) ; 5 2GA G A GA GA T A TC TTA A GC TGC CTT GCC A GA23 ( IGF2 ) ; 5 2GA G A GA CA T A TG GAA GTG GCT TCG 5 2GA G A GA GA T A TC TTA CTC TGA CTT PCR pbluescript SK EcoR, DNA, IGF cdna ( Shamblott et IGF2 IGF2 IGF2 IGF2 N - 21, 113 IGF2 2 h IGF2 1 h, ml 5 ml 100 mol/ L PMSF, g 30 min, zole, 015 mmol/ L NaCl, 20 mmol/ L Tris2HCl p H, 1 p H 719) mmol/ L Imidazole, IGF2 IGF2 His6 1 Elute buffer (1 mol/ L Imidazole, 015 mol/ L NaCl, 20 mmol/ L Tris2HCl p H 719) IGF2 IGF2 His6 48 h, Tricine2SDS2
3 IGF2 IGF2 His6 Fig11 The expression of rainbow trout IGF2 and IGF2 His6 fusion polypeptides M : (Lane M : protein molecular weight marker) 1 : (Lane 1 : pre induction of con2 trol) 2 4 : 1, 2, 3 h (Lane 2 4 : 1, 2, 3 hours post induction of control respectively) 5 : p ETIGF2 (Lane 5 : pre induction of p ETIGF2 ) 6 : 2 h p ETIGF2 (Lane 6 : 2 hours post induction of p ETIGF2 ) 7 : 2 h p ETIGF2 (Lane 7 : the supernatant of the cell lysate of the induced p ETIGF2 ) 8 : 2 h p ETIGF2 (Lane 8 : the pellet of the cell lysate of the induced p ETIGF2 ) 9 : p ETIGF2 (Lane 9 : pre induction of p ETIGF2 ) 10 : 1 h p ETIGF2 (Lane 10 : 1 hour post induction of p ETIGF2 ) 11 : 1 h p ETIGF2 (Lane 11 : the supernatant of the cell lysate of the induced p ETIGF2 ) 12 : 1 h IGF2 (Lane 12 : the cell lysate pellet of the induced p ETIGF2 ) IGF His6 ( The arrow indicates the band of rainbow trout IGF His6 fusion polypeptides) PA GE Tricine2SDS2PA GE Schag2 16 %, R2250, Gel Doc 1000 ( Bio2Rad) 96, :, 72 h, Molecular Analyst (Bio2Rad), Bio2Rad Protein Assay p ET215b BL21 (DE3) ( Bio2Rad), BSA, 115 BALB/ N IH3 T3 IGF 211 IGF2 IGF2 His6 Schirmer et al., Bisbenzimidazole ( H33258) CytoFluor TM (AB I) (360 nm, 460 nm ) 10 % FBS (bf GF) 100 g/ ml BSA 1 mol/ L ger et al. (1987), 4 %, (Dexamethasone) F12/ DMEM 4, 4 2 (1992) IGF2 IGF2 His6, BL21 (DE3) 1 p ETIGF2 p ETIGF2 BL21 (D E3) IPTG (014 mmol/ L) 2 h 1 h, F12/ DME BALB/ N IH3 T kda,, 5 IGF2 IGF2 His6 g/ ml ( Fibronectin) 10 g/ ml, 1010 kda ( Transferrin) 5 ng/ ml, IGF2 IGF2
4 2 : IGF2 IGF2 His6 Fig12 The purif ication of rainbow trout IGF2 and IGF2 His6 fusion polypeptides M : (Lane M : protein molecular weight marker) 1, 2 : (Lane 1 and 2 : the pre and post induction of control respectively) 3, 4 : p ETIGF2 (Lane 3 and 4 : the pre and post induction of p ETIGF2 respectively) 5 : IGF2 (Lane 5 : the column2 purified rainbow trout IGF2 ) 6, 7 : p ETIGF2 (Lane 6 and 7 : the pre and post induc2 tion of p ETIGF2 respectively) 8 : IGF2 (Lane 8 : the column2purified rainbow trout IGF2 ) 9 : (Lane 9 : the control after running through all purification steps as recombinant rainbow trout IGFs) IGF His6 ( The arrow indicates the band of rainbow trout IGF His6 fusion polypeptides) His6 His6, IGF2 212 IGF His6, 40 % 60 % His6 Ni 2 + 2His Bind 1 IGF2 IGF2 Binding buffer, 1 Wash buffer 100 mmol/ L Im2 idazole, 1 Elute buffer p ET215b, SDS2PA GE, Da, SDS2PA GE 2 p ETIGF2 p ETIGF kda, 1010 kda ;, IGF2,, IGF2 His6 80 %, 90 %, 100 g 173 g, IGF 213 IGF2 I IGF2 His6 IGF, 3 IGF2 IGF2 His6 IGF2 ( Moriyama et al., 1993 ) IGF2, (Upton et al., 1997) IGF2 (, BALB/ N IH3 T3, 2001),, 80 % 20 %,, 130 ng/ ml 260 ng/ ml : IGF2 IGF2 IGF2 3 IGF2 IGF2 B2C2A2D cdna IGF2 ( Fine et al., 1997),, IGF His6 6,
5 IGF2 IGF2 His6 BALB/ NIH3T3 Fig13 The mitogenic effects of rainbow trout IGF2 and IGF2 His6 fusion polypeptides on BALB/ NIH3T3 cells rhigf2 : IGF2 ( rhigf2 : recombinant human IGF2 ) rhigf2 : IGF2 ( rhigf2 : re2 combinant human IGF2 ) rrt IGF2 : IGF2 His6 ( rrt IGF2 : rainbow trout IGF2 His6 fusion polypeptide) rrt IGF2 : IGF2 His6 (rrt IGF2 : rainbow trout IGF2 His6 fusion polypeptide) rrt IGF2 0, 5614, 11218, 225, 450 ng/ ml ( The test concentrations for rrt IGF2 are 0, 5614, 11218, 225, and 450 ng/ ml respectively) ; rrt IGF2 0, 5617, 11215, 225, 450, 900 ng/ ml ( The test concentrations for rrt IGF2 are 0, 5617, 11215, 225, 450, and 900 ng/ ml respectively) (Values represent Means SD, n = 4) Ni 2 + 2His Bind, BALB/ N IH3 T3 IGF2,, IGF2, IGF2 : IGF2 (Reeth et al., 1998) IGF2 IGF2 His6 IGF2 IGF, (OπDell et al., 1998), IGF IGF2 IGF2 ( Mukhija et al., 1995 ; Tian et ( Humbel, 1990) IGF2 BALB/ al., 1999 ;, 2001) N IH3 T3 IGF2 : IGF2 IGF BALB/ N IH3 T3
6 2 : 271 IGF IGF ( Fine et al., 1997), His6,, IGF2 IGF2, IGF2 IGF2 U niversity of Con2 necticut T. T. Chen, Maria Chen IGF2 IGF2, ( References) Chen, J. Y., J. C. Chen, C. Y. Chang, S. C. Shen, M. S. Chen and J. L. Wu 2000 Expression of recombinant tilapia in2 sulin2like growth factor2 and stimulation of juvenile tilapia growth by injection of recombinant IGFs polypeptides. A quac. 181 : Duan, C The insulin2like growth factor system and its biologi2 cal actions in fish. A mer. Zool. 37 : Duan, C Nutritional and developmental regulation of insulin2 like growth factors in fish. J. N ut r. 128 : 306S 314S. Fine, M., R. Amuly, Y. Sandowski, T. A. Marchant, S. J. Chan, A. Gertler and B. Funkenstein 1997 Recombinant gilthead seabream ( S parus aurata ) insulin2like growth factor2 : sub2 cloning, expression in Escherichia coli, purification and characteri2 zation. J. Endocri nol. 153 : Hua, Y. M. and H. R. Lin 2001 Cloning and expression in prokaryote of grass carp IGF2 cdna. Acta. Zool. Si n. 47 : [, 2001 IGF2 cdna. 47 : ] Humbel, R. E Insulin2like growth factors and. Eur. J. Biochem. 190 : LeRoith, D., C. Bondy, S. Yakar, J. L. Liu and A. Butler 2001 The somatomedin hypothesis : Endocrine Reviews 22 (1) : Lindner, P., K. Bauer, A. Krebber, L. Nieba, E. Kremmer, C. Krebber, A. Honegger, B. Klinger, R. Mocikat and A. Pluck2 thun 1997 Specific detection of his2tagged proteins with recom2 binant anti2his tag scfv2phosphatase or scfv2phage fusions. Biotechniques 22 : Moriyama, S., S. J. Duguay, J. M. Conlon, C. Duan, W. W. Dickhoff and E. M. Plisetskaya Recombinant coho salmon insulin2like growth factor : expression in Escherichia col2 i, purification and characterization. Eur. J. Biochem. 218 : Mukhija, R., P. Rupa, D. Pillai and L. C. Garg 1995 High2level production and one2step purification of biologically active human growth hormone in Escherichia coli. Gene 165 : OπDell, S. D. and I. N. Day 1998 Insulin2like growth factor ( IGF2 ). Int. J. Biochem. Cell Biol. 30 : Reeth, T. V., P. L. Dreze, C. Szpirer and P. Gabant 1998 Posi2 tive selection vectors to generate fused genes for the expression of his2tagged proteins. Biotechniques 25 : Schagger, H. and G. von Jagow 1987 Tricine2sodium dodecyl sul2 fate2polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kda. A nal. Biochem. 166 : Schirmer, K., R. C. Ganassin, J. L. Brubacher and N. C. Bols 1994 A DNA fluorometric assay for measuring fish cell prolifera2 tion in microplates with different well sizes. J. Tissue Cult ure Methods 16 : Shamblott, M. J. and T. T. Chen 1992 Identification of a second insulin2like growth factor in a fish species. Proc. N atl. Acad. Sci. USA 89 : Tian, X. C., M. J. Chen, A. G. Pantschenko, T. J. Yang and T. T. Chen 1999 Recombinant E2peptides of pro2 GF2 have mitogenic activity. Endocri nology 140 : Upton, Z., G. L. Francis, S. J. Chan, D. F. Steiner, J. C. Wallace and F. J. Ballard 1997 Evolution of insulin2like growth factor ( IGF) function : production and characterization of recombinant hagfish IGF. Gen. Com p. Endocri nol. 105 : Yang, H., Y. Q. Zhang, Z. Yan, W. Han, L. B. Yao and C. Z. Su 2001 Human angiogenin : expression, purification, and bio2 logical assay. Chi nese Journal of Biotechnology 17 (1) : [,,,,, (1) : ] Ye, X., J. J. Bai, Q. Jian, X. H. Li, Y. H. Li, K. B. Li and J. R. Luo 2001 Expression of grass carp ( Ctenopharyngodon i dell us) insulin2like growth factor in E. coli. Chinese Journal of Biochemist ry and Molecular Biology 17 (6) : [,,,,,, (6) : ] Ye, X., J. J. Bai, H. H. Lao, Q. Jian, Y. H. Li and J. R. Luo 2002 Fusion expression and purification of Ctenopharyngodon i dell us IGF2 and preparation of the antiserum against GST2 GF. J. Fish Chi na 26 (2) : [,,,,, (2) : ] Zhang, Z. X. and Q. Z. Yao 2001 Fusion protein : a new technol2 ogy of biological engineering. Pharmaceutical Biotechnology 8 (1) : [, (1) : ]
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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