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1 2007, 29 (1 ) : Acta Botanica Yunnanica AOX) 1, 3, Moore A L 2, 3, 3 (1, ; 2 School of Biological Sciences, University of Sussex, BN1 9QG UK; 3, ) : ( Alternative Oxidase, AOX), Q, 4 AOX AOX, pflag-1, - -D- ( IPTG) AOX, pflag-1-aox : DH5 37 OD 600 = 0.6 IPTG 0.2 mmol L, 60 min ; AOX AOX, AOX, : ( AOX) ; pflag-1 ; : Q 945 : A : (2007) Optimal Expression of Alternative Oxidase in Escherichia coli WANG Ya-Ying 1, 3, MOORE A L 2, YANG Shen-Chang 3, TIAN Hui-Qiao 3 ** ( 1 Agricu ltural Institute, Zhangzhou , China ; 2 School of Biological Sciences, University of Sussex, UK BN1 9QG; 3 School of Life Sciences, University of Xi amen, Xiamen , China) Abstract : Alternative oxidase ( AOX), a terminal oxidase located in the inner mitochondrial membrane, has been identified universally in plant, algae, fungi and protozoan. AOX branches from the cytochrome pathway at the level of the ubiquinone and catalyses the oxidation of ubiquinol and reduction of oxygen to water. The enzyme remains difficult to purify to homogeneity in a stable, active form. The paper expressed the AOX with inducer IPTG in pflag-1 Escherichia coli system. pflag-1 carry a ompa which secretary the AOX to periplasm. Three strategies have combined to increase the yield of AOX. First strategy involved tuning of the inducer concentration while the second involved alter the growth temperature and the last involved differ the cell concentration. The optimized protocol is that the host cell is DH5, the temperature is 37, the cell concentration is OD 600 = 0.6 and the IPTG is 0.2 mmol L. Purification of proteins showed AOX is expressed in unsoluble protein. Key words: AOX; pflag-1 Escherichia coli Expression System; Over-expression (AOX) Q, AOX, ATP, (Moore, 1978), AOX, AOX, (, 2003 ), (, 1999), (, 2004 ) (, 2003) AOX,, : ( ) : Author for correspondence ; hqtian@ jingxian. xmu. edu. cn; Tel: : , : ( ),,,

2 mmol L, 60 min ; AOX AOX, AOX, : ( AOX) ; pflag-1 ; : Q 945 : A : 0253-2700 (2007) 01-074 - 05 Optimal Expression of Alternative Oxidase in Escherichia coli WANG Ya-Ying 1, 3, MOORE A L 2, YANG

2 1 : (AOX) 75 AOX, AOX (Affourtit, 2002) AOX, AOX AOX, AOX,, AOX AOX ( Berthold, 2002; Albury, 2002 ), AOX ( Affourtit, 1999 ), AOX,,, AOX C41, C42 ( Berthold, 2003 ), pflag-1, AOX, AOX, AOX ( Sambrook and Russell, 2003 ), AOX, AOX,, IPTG, (OD 600 ), AOX ( Sigma, 1998 ) pflag-1 AOX, AOX ( Subcloning Efficiency DH5, Chemically Competent E. coli) Invitrogen; pflag-1 ; Western Blotting (Kit ECL for AOX detection) SIGMA ; ( WM) RAN- BOW RPN800 Amersham Biosciences ; pflag-1-aox, AOX, SUSSEX Moore ; AOX ( Sauromattum guttatum) pflag-1-aox pflag-1 (Ampicillin, Amp) puc19 ddh 2 O, 50 g ml Amp LB 4 DH5 50 L, pflag-1-aox pflag-1 puc19 ddh 2 O 3 L, 30 min, s, 2 min, 90 L LB, 100 l LB (Amp50 g. 50 ml - 1 ), AOX pflag-1-aox pflag-1 ( cells ) 5 ml LB h, 0.5 ml 50 ml LB, OD , IPTG 0.5 mmol L AOX, IPTG 60 min 90 min 120 min 1 ml OD 600, 1 ml, r min 1 min, , 90 5 min,, 10 % SDS-PAGE, Western blotting AOX ( Albury, 1996) AOX 3 OD 600 = 0.2, 0.6, 1.0, 3 IPTG 0.05 mmol L, 0.2mmol L, 0.5 mmol L, 37 AOX (Sigma, 1998), : (1) 100 ml (a) ( b), min ( r min) ; 2 ml - 20, Western blot AOX; (2) (a) 0.4 g, 16 ml (500 mmol L Sucrose, 30 mmol L Tris-HCl, 1 mmol L EDTA ), : 1 g. 40ml - 1 ; min) ; ; (3) 10, 10 min (5 500 r (4) 12 ml 4, ; (5) 4 10 min (5 500 r min), 2 ml - 20, Western blot AOX; (6) (b) 5 ml A (50 mmol L Tris-HCl, 5 mmol L EDTA ; 0.25 mg ml lysozyme; 50 ug ml sodium azide, ph8), 5 min, ; (7) B 0.5 ml (1.5 mol L NaCl, 0.1 mol L CaCl 2, 0.1 mol L MgCl 2 ), 5 min; (8 ) 30 min ( r min), 2 ml - 20, Western blot AOX; (9) 5 ml A, 2 ml - 20, Western blot AOX AOX AOX ANTI-FLAG. M1 (0.2 ml) (Sigma, 1998) : AOX (1 ) 1 ml TBS (50 mmol L Tris-HCl, 150 mmol L NaCl, ph7.4), 400 L

coli) Invitrogen; pflag-1 ; Western Blotting (Kit ECL for AOX detection) SIGMA ; ( WM) RAN- BOW RPN800 Amersham Biosciences ; pflag-1-aox, AOX, SUSSEX Moore ; AOX ( Sauromattum guttatum) 1.2

3 76 29 AOX FLAG ; (2) 1.0 ml 0.1 mol L 3, 1.0 ml TBS 3 ; (3) 0.5 ml 0.5 ml 2 TBS 0.1 mol L CaCl 2,, 3, AOX ; (4) 0.2 mmol L EDTA TBS 6, AOX, ( E1-E6), - 20, Western blot AOX (TCA deoxycholate) AOX, Western blot TCA 20 1 ml 0.2 ml Sodium deoxycholate 0.2 ml 10 min ( r min),, 5 % ( w v ) SDS 50 L, Western blot AOX tac pflag-1 ( 1) 8 ( FLAG-1 ), IPTG AOX FLAG 8 FLAG, AOX, FLAG AOX 2.2 AOX 2 2.1,, pflag-1 pflag-1-aox puc19, ddh 2 O, pflag-1-aox pfla-1g puc19 ( amp r ), 50 g mlamp 1 pflag ompa. ; FLAG. FLAG8 ; amp r. ; lac. Fig. 1 Expression vector of pflag ompa: Signal sequence for secretion of FLAG fusion proteins to perplasmic space; FLAG: Octapeptice for binding of Anti-FLAG antibodies; amp r : Ampicillin resistance to host cell; tac : - 35 region of trp promoter to end of laci binding region; laci: repression of tac promoter. 2B AOX 2A Western Blot AOX Fig. 2A The AOX fusion protein showed by Western Blot with Anti-AOX antibodies 2, 4: pflag-aox + IPTG ( AOX fusion protein induced by IPTG) ; 1, 3: pflag-aox-iptg ( no AOX protein induced by without IPTG; 5 : the AOX isolated from Arum maculatum mitochondrials ; , 75: RPN800 markers Fig. 2B Comparing the growth rat of the host cells between AOX expressed and no AOX expressed pflag + IPTG ( blue) : no AOX gene induced by IPTG to produce no AOX; pflag-iptg ( red ) : no AOX gene and no IPTG to produce no AOX; pflag-aox-iptg ( green ) : AOX gene induced by no IPTG to produce no AOX; pflag-aox + IPTG ( yellow) : AOX gene induced by IPTG to produce AOX.

1,, pflag-1 pflag-1-aox puc19, ddh 2 O, pflag-1-aox pfla-1g puc19 ( amp r ), 50 g mlamp 1 pflag ompa. ; FLAG. FLAG8 ; amp r. ; lac. Fig.

4 1 : (AOX) 77 : 37, OD 600 = 0.2, IPTG 0.5 mmol L, Western Blot AOX, ( 2A) AOX,, 60 min ( 2B), AOX, ( Sambrook and Russell, 2003),,, ( 3), OD 600 = 0.6, IPTG 0.2 mmol L, 37 AOX, 4 h, OD Western blot, AOX ( 4) AOX 32 kda 70 kda, AOX 35 kda 70 kda 2 (, 2002) AOX, 2 : kda kda IPTG 0.2 mmol L, 3, AOX, OD 600 = 0.6, 3 IPTG AOX 30 min 120 min AOX, AOX,, h ( ), AOX 1 h,, (, 2003) AOX Berthold (2002), 18, OD 600 = 0.5 IPTG 0.1 mmol L, 10 h AOX : IPTG 0.2 mmol L, OD 600 = 0.6, 37, 60 min 3 37, IPTG = 0.05, 0.2, 0.5 mmol L, OD = 0.2, 0.6, 1.0 Fig. 3 Growth of bacterial with AOX expression induced under 37 and different IPTG concentration with different density of cell 4 37, 0.2 mmol L IPTG, AOX ( Western blot) Fig. 4 Optimal expression AOX induced by IPTG 0.2 mmol L under 37 and different cell density 0: befoure induced by IPTG; 30, 60, 120: The time after induced by IPTG ( min) ; Ar: mitochondria isolated from Arum maculatuml; : OD 600 = 0.2, 0.6, AOX ( Western blot) Fig. 5 Purified & concentrated AOX from periplasm 1, 3, 5, 7 : the periplasm AOX induced under 37, 30, 25, 18 ; 2, 4, 6, 8: the unsoluble AOX induced under 37, 30, 25, 18 ; 9: the AOX of mitochondrial isolated from Arum maculatum.

6, 3 IPTG AOX 30 min 120 min AOX, AOX,, 18 10 h ( ), AOX 1 h,, (, 2003) AOX Berthold (2002), 18, OD 600 = 0.5 IPTG 0.1 mmol L, 10 h AOX : IPTG 0.2 mmol L, OD 600 = 0.6, 37, 60 min 3 37, IPTG = 0.

5 78 29 ( 3 ) [ M]. :, AOX, AOX, AOX,, AOX, pfalg- 1-AOX AOX AOX ( Berthold, 2002, 2003), AOX 37 TAO, 30 TAO 80% TAO TAO, TAO TAO (Nihei, 2003) ( Sambrook and Russell, 2003 ) , AOX 20, AOX, 37 AOX ( 5 ), AOX, 3, AOX, AOX AOX, AOX,, AOX, pflag-1-aox, : 37, OD 600 = 0.6, IPTG 0.2 mmol L AOX, AOX AOX, AOX, AOX Sambrook J, Russell DW, ( ), Affourtit C, Albury MS, Krab K et al Functional expression of t he plant alte rnative oxidase affects growth of the yeast Schizosaccharomyces pombe [ J]. J Biol Chem, 274 ( 10) : Affourtit C, Albury MS, Crichton PG et al Exploring the molecular nature of alternative oxidase regulation and catalysis [ J ]. FEBS Lett, 510: Albury MS, Dudley P, Watts FZ et al Targeting the plant alternative oxidase protein [ J ]. J Biol Chem, 271: Albury MS, Affourtit C, Crichton PG et al Structure of the plant alternative oxidase : site- directed mutagenesis provides new information on the active site and membrane topology [ J ]. J Biol Chem, 277: Bert hold DA, Voevodskaya N, Stenmark P et al EPR studies of the mitochondrial alternative oxidase-evidence for a diiron carboxylate center [ J]. J Biol Chem, 277: Bert hold DA, Stenmark P, Nordlund P et al Screening for functional expression and overexpression of a family of diiron-containing interfacial membrane proteins using the univector recombination system [ J ]. Protein Sci, 12 : He QX ( ), Wei ZQ ( ), Liang HG ( ), Effects of water stress on the Cyanide-resistan t respiration and expression of the alternative oxidase gene in wheat seedlings [ J ]. Sci Chin ( Ser C) ( C ), 29 (4 ) : Liu GL ( ), Zhang XX ( ), Zhang XZ ( ), Cloning and prokaryotic expression of ubiquitin gene of spodoptera exigua multi-nucleocapsid nucleopolyhedrovirus [ J ]. Virol Sin ( ), 18 (1 ) : FLAG E. coli Expression System Manual [ M]. Sigma, Moor e AL, Bonner WD, Rich PR et al Determination of protonmotive force during cyanide- insensitive respiration in plant mitochondria [ J ]. Arch Biochem Biophys, 186: Nihei C, Fukai Y, Kawai K, 2003a. Purification of active recombinant trypanosome alternative oxidase [ J ]. FEBS Letters, 538: Wu Q ( ), Li HY ( ), Zhang LX ( ) et al The Stress and the cyanide-resistant respiration [ J ]. Acta Bot Boreal-Occident Sin ( ), 23 (1) : Yan YC ( ), Lin HH ( ), Lian HG ( ) et al Comparison of the effects of different low temperature stresses on the induction of the cyanide-resistant alternative pathway and the expression of alternative oxidase in Tobacco callus [ J ]. Chin Bull Bot ( ), 21 (3 ) : Zhang WL ( ), Shen WB ( ), Ye MB ( ) et al Aconitase and its physiological rolesin plants [ J ]. Plant Physiol Commun ( ), 39 (4) :

), AOX, 3, AOX, AOX AOX, AOX,, AOX, pflag-1-aox, : 37, OD 600 = 0.6, IPTG 0.2 mmol L AOX, AOX AOX, AOX, AOX Sambrook J, Russell DW, ( ), 2003. Affourtit C, Albury MS, Krab K et al. 1999.

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untitled 20 1 2010 10 Vol.20 Special 1 The Chinese Journal of Nonferrous Metals Oct. 2010 1004-0609(2010)S1-s0127-05 Ti-6Al-4V 1 2 2 (1. 710016 2., 710049) 500~1 000 20 Ti-6Al-4V(TC4) TC4 800 TC4 800 TC4 TC4 800