49 (2) :266 271, 2003 A cta Zoologica S inica 3 33 (, 510275) 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 510275, 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) : 266-271, 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), 2002204215, 2002211228 3 (No1990253) (2000078314301) [ This study was funded by Natural Science Foundation of Guangdong Province (No1990253) and State Programme for Skeleton Teachers in Higher Institution ] 33 (Corresponding author) E2mail : ls68 @zsu1edu1cn,, 36,,, : ν 2003 Acta Zoologica Sinica
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 600 2001) IGF2 IGF2 019, 4, His6, 100 ml LB ( Ampicilin 75 g/ ml) IGF2 IGF2 His6, 37 OD 600 019, IPTG, BALB/ 014 mmol/ L N IH3 T3 1 111 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, - 20 114 100 ml 5 ml 100 mol/ L PMSF, 4 20 000 g 30 min, zole, 015 mmol/ L NaCl, 20 mmol/ L Tris2HCl p H, 1 p H 719) 3 100 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
68 2 49 1 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, 5 000 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 T3 1010 kda,, 5 IGF2 IGF2 His6 g/ ml ( Fibronectin) 10 g/ ml, 1010 kda ( Transferrin) 5 ng/ ml, IGF2 IGF2
2 : 269 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, 9 90917 9 47419 Da, SDS2PA GE 2 p ETIGF2 p ETIGF2 1010 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,
70 2 49 3 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
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