Purif ication of Ovalbumin from Hen Egg White by High- speed Counter-current Aqueous Two- phase Chromatography

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1 Chinese Journal of Biotechnology Vol. 21 No. 1 January 2005 Purif ication of Ovalbumin from Hen Egg White by High- speed Counter-current Aqueous Two- phase Chromatography 1 3, 2, 1 1, ZHI Wen-Bo 1 3, DENG Qiu- Yun 2, SONGJiang-Nan 1 and OUYANG Fan 1 11, , State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, CAS, Beijing , China 21 TAUTO Biotech Co., LTD, Shanghai , China -, Π PEG1000- ph PEG ph % ( WΠW) PEG % ( WΠW) C, 018mLΠmin,850rΠmin, C ph % ( WΠW) PEG % ( WΠW), :, 118mLΠmin 850rΠmin,200 min, 100 %, 95 %,,,, TQ93 A (2005) Abstract High- speed counte-rcurrent chromatography ( HSCCC) is a continuous liquid-liquid partition chromatography without solid matrix, which has the significant features of high resolution and high recovery. The separation of bio- macromolecule in aqueous two-phase systems ( ATPs) with HSCCC is still under research, and the establishment of high- speed counter- current aqueous two-phase chromatography ( HSCCC-ATP) relies on the improvement of equipment structure and optimization of operation parameters. By using a multi- column high- speed counter- current chromatograph, the separation of protein mixture and the purifi2 cation of ovalbumin from hen egg white were studied. The effects of ph and PEG concentration on the partition coefficients of proteins were tested in PEG1000-phosphate ATPs, and distinct differences among partition coefficients of proteins were found at ph 912 and 1510 % ( WΠW) PEG concentration in said system. The separation of protein mixture, consisting of cytochrome C, lysozyme and myoglobin was successfully performed in 1510 % ( WΠW) PEG % ( WΠW) potassium phosphate ATPs at ph 912 with high- speed counter- current chromatograph at rotation speed of 850rΠmin and flow rate of 018mLΠmin, using upper Received : August 9, 2004 ; Accepted : October 12, Corresponding author. Tel : ;

2 130 Chinese Journal of Biotechnology 2005,Vol121,No11 phase as stationary phase. ph and PEG concentration also had distinct effects on the partition coefficients of the major protein components in hen egg white, including ovaltransferrin, ovalbumin and lysozyme. The optimal ph value and PEG concentration for the purification of ovalbumin by HSCCC-ATP were found to be 912 and 1610 % ( WΠW) respectively. Ovalbumin was suc2 cessfully purified to homogeneity from the hen egg white sample in 1610 % ( WΠW) PEG % ( WΠW) potassium phos2 phate ATPs at ph 912 with high- speed counter- current chromatograph at rotation speed of 850rΠmin and flow rate of 118mLΠmin, using upper phase as stationary phase. The purification recovery of ovalbumin was around 95 %. Key words high- speed counter-current chromatography, aqueous two-phase system, purification, protein, ovalbumin ( High Speed Counter- current Chro2 matography, HSCCC) 2 [1,2 ] 111 TBE-300V (, ) ( 216mm), 1,3 Π, 120mL ( 1 ), [3,4 ], (HX-1050, ) 20mL ( 70 % 90 %) rΠmin [5 ], KTA TM PRIME (Amersham), [6,7 ],, [5 ] [4 ], HSCCC 1 TBE-300V Fig. 1 Diagram of high speed countercurrent chromatography [8,9 ] model TBE-300V, [10,11 ], 1 A : KTA TM prime ; B : TBE-300V ; C: water bath ; 1, 2 and 3 : separation columns. HSCCC TBE- 300V 112, PEG1000 ( ) ; Π ( ) ;, : C ( ) C ( ) ( ) ( ) ( ) ( ) Sigma (Sigma, USA), :,,, ( ), B

3 : 131 (97kD) (66kD) (43kD) 10 %, (31kD) (20kD) 1110 (14kD) 113 PEG 1Π2, mg 2mL,, 015mL, C, 280nm, K = A U ΠA L 115 PEG : 1mg C, ph PEG C 5mg 20mg 2mL ph PEG1000, 1310 % ( WΠW) PEG % ( WΠ : Awad [12 ] W) ph912 PEG10002 : 2 014molΠL NaCl 10mmolΠL molΠL Tris- HCl (ph 910) 5mL, PEG1000, 116 HSCCC,, ; ; 117 HSCCC,, ; Ve ( R) ( Vt), R = ( Vt - Ve)Π( Vt) 118 Bradford [13 ] 1mgΠmL (BSA) 2 ph (A) PEG (B),, 595nm Fig. 2 Effect of ph (A) and PEG concentration (B) of aqueous two-phase system on the partition coefficients of proteins 119 ( ) lysozyme ; ( ) myoglobin ; ( ) cytochrome C. [14 ] SDS- PAGE BandScan (V4130,Glyko) 2 2 ph PEG 2

4 132 Chinese Journal of Biotechnology 2005,Vol121,No11 2 C ( ), ph PEG10002 ph PEG ; ph 8, ph912 PEG PEG ; ph, 110, ph912 PEG10002 PEG ph PEG ph % ( WΠW) PEG PEG10002,,, : :216mm 120mL ; :1mg C,8mg 20mg ; : PEG = ( %, WΠW) ; : ( PEG) ; : ( ) ; :018mLΠmin ; :850rΠmin ; :40 %, 3 4 ph (A) PEG (B) Fig. 4 Effect of ph (A) and PEG concentration (B) of aqueous two-phase system on the partition coefficients of protein components in hen egg white ( ) lysozyme ; ( g ) ovalbumin ; ( ) ovaltransferrin. PEG ph PEG Fig. 3 HSCCC chromatogram of mixture of cytochrome C, myoglobin and lysozyme [15 ] ph 912 PEG 1 :cytochrome C; 2 :myoglobin ; 3 : lysozyme. ( 1410 %, WΠW) PEG10002 ( C, ), 3, 213 PEG10002, ph912 PEG10002 (Rs) (Sigma , 40 % ) 212 ph PEG : :216mm ( 78kD) ( 45kD) (14kD) 120mL ; :150mg (Sigma) ;

5 : 133 : PEG = ( %, WΠW) ; : ( 5),, PEG) ; : ( ) ; [12 ] S :112 mlπmin ; :850rΠmin ; :, 38 % 5, 5mL, ( ) 118mLΠmin, ; ( 7, 6) 35 %, 6,, (, ) 118mLΠmin, 200min ( 7), 35 %, 3) ( 7, BandScan 17 %, 55 %, 10 % 240mg 132mg 2 78mg, 3 48mg, 95 % 5 Fig. 5 HSCCC chromatogram of hen egg white powder from Sigma 5, ( ),,, 6, Fig. 6 HSCCC chromatogram of hen egg white sample 7 Fig. 7 SDS- PAGE result of elution peaks of hen egg white sample after high- speed countercurrent chromatographic separation 1 : protein marker ; 2 : hen egg white sample ; 3 : peak 1 ; 4 : peak 2 ; 5 : peak 3 ; 6 : upper stationary phase. 3 ph PEG ( TBE-300V) 6, ( 1) ph % ( 60 %, 7, 3), ( WΠW) PEG % ( WΠW), ( 2) ( 7 018mLΠmin 850rΠmin 4) ( C

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