GB2498705A - Recombinant Alternative Oxidase - Google Patents

Abstract

The invention provides genetic constructs comprising a coding sequence encoding recombinant alternative oxidase (rAOX), and extends to a method of producing highly active and pure recombinant alternative oxidase, and contacting the rAOX with a keto-acid.

Description

ALTERNATIVE OXIDASE

The present invention relates to alternative oxidases, and in particular to plant, fungal and bacterial recombinant alternative oxidases. The invention is concerned with genetic constructs comprising a coding sequence encoding alternative oxidase, and extends to a method of producing highly active and pure recombinant alternative oxidase.

The alternative o)udase @OX) is a non-proton motive uhiquinol oxido-reductase, which catalyzes the 4-electron reduction of dioxygen to water. Genes encoding AOX have been found in higher plants, algae, fungi, yeast, slime molds, free-living amoebae, eubacteria, nematodes and protists. Moreover, recent hioinformatic searches have broadened the taxonomic distribution of AOX to some members of the animal kingdom. The primary roles of AOX in non-therniogenic plants include the regulation of the cellular redox balance and the protection of cells from reactive oxygen species, specifically when the cytochrome pathway is inhibited or when the respiratory chain has been impaired. However, AOKs also play a role in the metabolism of a multitude of other organisms. Its ubiquitous nature may suggest that the metabolic ilexihility that the alternative pathway confers upon an organism permits it to respond to a wide range of developmental and environmental conditions.

Until recently, it has not been possible to characterize the structural features of the alternative oxidase family. Although, to date, no high resolution structures of plant AOX have been determined, current structural models predict that AOX contains a non-haem diiron carhoxylate active site and is an integral, interfacial membrane protein that interacts with a single leaflet of the lipid hilayer. These predictions are based on homology modelling data of the AOX enzyme, and this model is supported by extensive site-directed mutagenesis studies.

Furthermore, both Electron Paraniagnetic Resonance (EPR and Fourier Transform Infrared (FTIR) spectroscopy have confirmed the presence of a binuclear iron center in both the plant and trypanosonial enzymes.

There is, however, still a significant need to perform detailed structural and biochemical analyses of AOXs, and especially plant and fungal AOXs, and this is currently not possible due to a lack of suitable purification protocols which enable the production of sufficient amounts of punfied and highly active plant AOX, to enable crystallization trials and kinetic analyses to be conducted.

As described in the Examples, the inventors have now devised a novel method by which highly active and very stable recombinant AOX can be prepared.

Thus, in a first aspect of the invention, there is provided a method of preparing recombinant Alternative Oxidase rAOX9, the method comprising culturing a host cell comprising a genetic construct comprising a nucleic acid molecule which encodes an alternative oxidase AOX) under conditions suitable for the production of rAOX; and contacting the rAOX with a keto acid.

Advantageously, the method of the invention enables the preparation of purified rAOX, which is highly active and exhibits exceptional stabilit upon storage. Furthermore, kinetic characterization of the rAOX has revealed that it exhibits typical Michaehs-Menten kinetics and is potently inhibited both by ascofuranone and its derivative (colletochlorin B). The inventors have surprisingly shown that the presence of, and interaction with, a keto acid is important for the preparation of highly active rAOX, because it causes a conformational change in the enzyme, which results in a surprising increase in the overall activity of the enzyme. This was totally unexpected. Furthermore, the inventors have shown that a keto acid, such as pyruvate, also stahilises the rAOX, though the mechanism by which this is achieved is not fully understood.

It will be appreciated that a keto acid (also known as an oxoacid) can be an organic acid which contains a carhoxyhc acid group and a ketone group. In some cases, the keto group may he hydrated. The keto acid may he an alpha-keto acid (i.e. 2-oxoacid), a heta-keto acid (i.e. 3-oxoacid) or a gamma-keto acid @.e. 4-oxoacid). however, it is preferred that the keto acid is an alpha-keto acid. For example, the keto acid maybe pyruvic acid (i.e. pyruvate), oxaloacetic acid or glycolic acid. Preferably, the keto acid is pyruvate.

The concentration of keto acid contacted with the rAOX may be at least lmNi, 2mM, Smtvl or 8mM. Preferably, the concentration of keto acid is 10mM or less. Thus, the concentration of keto acid may he between I mM and I 0mM, or between 2mM and I 0mM, or between 5mM and 10mM.

The keto acid may form part of a buffered medium. For example, the medium may comprise Tris-J-ICI. The p1-I of the buffered medium may be between 7.0 and 8.0.

The method may comprise contacting the rAOX with an emulsifying agent. The rAOX may be contacted with the emulsifying agent after it has been contacted with the keto acid. A suitable emulsifying agent may be EDT-20. The concentration of the emulsifying agent may be at least 0.01% (v/v), 0.02?/ (v/v) or at least 0.025% (v/v). Preferably, the concentration of emulsifying agent is 0.025% v/v) or less. Thus, the concentration of emulsifying agent may he between 0.01% and 0.025% v/v, or between 0.02% and 0.025% v/v. The inclusion of an emulsifying agent, such as EDT-20, stabillses tile rAOX in its active-form, which the inventors believe maybe as a result of mimicking the mitochondrial membrane. /5

The host cell may be a yeast cell, a fungal cell or a bacterial cell, for example B. co/i. In one embodiment, the host cell may be a haem-deficient strain, for example B. co/i ShcYIL4 mutant (EN 102) strain, wInch lacks quinol oxidase activity of the cytochr)me ho and ha' complexes.

Advantageously, since this strain of 1-. co/i lacks some enzymes that would normally allow the B. co/i to grow, the expression of die rAOX rescues the host organism by allowing it to synthesise a terminal oxidase, thereby enabling the functional expression of AOX separate from other terminal oxidases. Alternatively, the host cell may be an animal cell, for example a mouse or rat cell. It is preferred that the host cell is not a human cell.

For example, in embodiments where the host cell is B. co/i, the method may comprise culturing the host cell in S-broth, which will he known to the skilled person. The culturing may comprise incubation at about 30°C until the OD6oo = approximately 0.1. Once the culture has reached optimum cell density, the method may comprise inducing the cells with a suitable inducing agent which is capable of stimulating expression of die rAOX. In one embodiment, the inducing agent may be lsopropyl--I)-thio-gaiactoside ([PTG). The optimum concentration of the inducing agent maybe at least 10pM, 2OpM, 3OpM, or 4OpM.

The concentration of the inducing agent may he 5OjJM or less. After induction with the inducing agent, the method may comprise incubating the host cell for at least a further 2, 4, 6, 8, 10, 12 or 14 hours to allow expression of the rAOX.

Following the incubation in the presence of the inducing agent, the host cell may be harvested, fhr example by centrifugation. The method may then comprise re-suspending harvested cell pellets in the presence of the keto acid, such as pyruvate. The method may comprise contacting the host cell with a protease inhibitor. For example, a suitable protease inhibitor cocktail may be that which is known as Complete' protease inhibitor cocktail tablets, available from Roche Diagnostic GrnbH, Germany. The method may then comprise lysing the host cell to release the rAOX. After lysis, cell debris may he removed, for example by centrifugation. Pellets containing the cell membranes may then he re-suspended in the presence of the keto acid, fbr example pyruvate.

The method may comprise soluhilising the host cell's cell membrane. Soluhilisation may comprise contacting the host cell with a suitable solubili2ation buffer, which may comprise the keto acid, for example pyruvate. The soluhilisation buffer may comprise n-Dodecyl-j]-D-maltopyranoside (DDM.

The method may comprise purifying the rAOX. Purification may comprise the use of chromatography, for example affinity chromatography. In some embodiments, the rAOX may comprise a histidine tag. Thus, in such embodiment in which rSgAOX was fused with N-terminal histidine tag, soluhilized rSgAOX may he purified by cobalt affinity chromatography. The rAOX which has been solubilized by 0DM maybe bound to an affinity resin in the presence of DDM. The rAOX bound to the resin may be eluted with buffer containing DDM. Purified rAOX may he obtained by elution with imidazole resulting in a very efficient purification of active enzyme.

The AOX maybe a eukaryotic or prokaryotic ACK enzyme. The AOX maybe a plant AOX, a fungal AOX or a bacterial AOX. The nucleic acid sequence encoding AOX enzymes may be found from the publicly available databases. For example, as described in the Examples, in one embodiment, the AOX may he Sauromaium gui/a/urn AOX, wInch is also referred to as I'.

venomrn. The DNA sequence encoding S. gui/a/mn AOX (Accession Number: M60330) is provided herein as SEQ TI) No: I, as follows: atgatga gctcgcgtct ggtcggcacc gctctctgca ggcagctcag tcacgtcccc gtacctcagt acttgcctgc cctccgtccc acggcggaca cggcgagctc actcctgcac ggatgttcag cggcggcgcc ggcgcagaga gcgggcctct ggccgcccag ctggttctog cccccccgcc acgcgagcac gctgtcagct coogcccagg acggagggaa ggagaaggct gcaggaacag eegggaaggt gceqeegggt gaggacggcg gegeegagaa ggaggcggtg gtgagctact gggcggtgcc gccgtccaag gtcagcaaag aggacggctc cgagtggcgc tggacctgct tcaggocatg ggagacgtac caggcggacc tctccatcga cctgcacaag caccacgtcc ccaccaccat tctcgacaag ctggccttgc gcaccgtcaa ggccctccgg tggcccaccg acatcttctt ccaqcggcgg taccjcatgcc gggcgatgat gctggagacg gtggcggcgg tgccgggcat ggtgggcggg gtactcctcc acctcaagtc cctccgccgc ttcgagcaca gcggcgggtg gatcagggcc ctcctggacjçj aggccgagaa cgagcggatg cacctgatga ccttcatgga ggtggcgcag ccgcggtggt acgagcgggc gctggtgctg gcggtgcagg gggtcttctt caacgcctac ttcctggggt acctgctctc ccccaagttc gcccaccggg ttgtgggcta cctggaggag gaggccatcc actcctacac cgagtteetc aaqqaeateg aeagtggqqc cateeaggae tgccccqccc eggeeategc cctggactac tggcggctgc cgcagggctc caccctgcgc gacgtcgtca ccgtcgtccg cgcagacgag gcacaccacc gcgacgtcaa ccacttcgcc toogacgtcc attaccagga tettgagetg aagacgacge eggegccgct cgggtaeeae tga [SEQ Ti) No: 1] 2 Therefbre, in one embodiment, the nucleic acid molecule, which encodes the rAOX, may comprise a nucleotide sequence substantially as set out in SEQ TD No:l, or a functional variant or a fragment thereof.

The polypeptide sequence of S. guttatum AOXis provided herein as SEQ TI) No: 2, as follows.

MMSSRLVGTALCRQLSHVPVPQYLPALRPTADTASSLLHGCSAAAPAQRAGUPPPSWFSPPRHASTLSAPAQDG G

KEKAAGTAGKVPPGEDGGAEKEAX/VSYWAVPPSKVSKEDGSEWRWTCFRPWETYQADLS IDLHKUHVPITILDKL ALRTVKALRWPTDI FFQRRYACRPaILETVAAVPGF4VGGVLLHLKSLRRFEHSGGW I RALLEEAENERMHLMTFF4 3 EVAQPRWYERuLVLAVQGVFFNAYFLGYLLSPKFAHRV\JGYLEEEAIHSYTEFLKDIDSGAIQDCPAPAIALD YW RLPQGSTLRDWJTWRADEAHHRDVNHFASDVHYQDLELKTTPAPLGYH* [SEQ TD No: 2] Therefore, in one embodiment, the rAOX may comprise an amino acid sequence substantially as set out in SEQ TD No: 2, or a functional variant or fragment thereof.

Genetic constructs used in the method of the first aspect may be in the form of an expression cassette, which may be suitable for expression of the rAOX in the host cell. The genetic construct may he introduced into the host cell without it being incorporated in a vector. For instance, the genetic construct, which may he a nucleic acid molecule, may he incorporated within a liposome or a virus particle. Alternatively, a purif9ed nucleic acid molecule (e.g. histone-free DNA, or naked DNA may be inserted directly into a host cell by suitable means, e.g. direct endocytotic uptake. Suitable means for introducing the genetic construct into die host cell will depend on the type of cell. The genetic construct may he introduced directly in to cells of the host subject (e.g. a bacterial cell) by transfection, infection, electroporation, microinjection, cell fusion, protoplast fusion or ballistic bombardment. Alternatively, genetic constructs of the invention may be introduced directly into a host cell using a particle gun.

Alternatively, the genetic construct may he harboured within a recombinant vector, for expression in a suitable host cell. The recombinant vector may he a plasmid, cosmid or phage.

Such recombinant vectors are useful for transforming host cells with the genetic construct, and for replicating the expression cassette therein, such that the rAOK is expressed. The skilled technician will appreciate that genetic constructs of the invention may be combined with many types of backbone vector for expression purposes. Examples of suitable backbone vectors include pET-i 5h (see Figure 2) to form an expression vector pETSgAOX (see Figure 3).

Recombinant vectors may include a variety of other functional elements including a suitable promoter to initiate gene expression and/or a suitable terminator to cease gene expression.

One example of a suitable promoter may he the T7 promoter, and an example of a suitable terminator may he the T7 terminator. The recombinant vector may he designed such that it autonomously replicates in the cytosol of the host cell. In this case, elements which induce or regulate DNA replication may he required in the recombinant vector. Alternatively, the recombinant vector may he designed such that it integrates into the genome ofa host cell. In this case, DNA secluences which favour targeted integration (e.g. by homologous recombinanon) are envisaged.

The recombinant vector may also comprise DNA coding for a gene that may be used as a selectable marker in the cloning process, i.e. to enable selection of cells that have been transfected or transformed, and to enable the selection of cells harhouring vectors incorporating heterologous DNA. For example, ampicillin resistance is envisaged.

Alternatively, the selectable marker gene may he in a different vector to he used simultaneously with vector containing the gene of interest. The vector may also comprise DNA involved with regulating expression of the coding sequence, or for targeting the expressed polypeptide to a certain part of die host cell.

In a second aspect, there is provided a genetic construct substantially as represented in Figure 3.

The construct may comprise a nucleotide sequence substantially as set out in SEQ ID No:l, or a functional variant or a fragment thereof and/or encode a rAOX, which comprises an amino acid sequence substantially as set out in SEQ TI) No: 2, or a functional variant or fragment thereof.

In a third aspect, there is provided recombinant Alternative Oxidase rAOX obtained by, or obtainable from, the method of the first aspect.

The rAOX of the third aspect may be stable for at least 1, 2, 3, 4, 5 or 6 months.

The activity of the rAOX may he greater than lOIJMol, 2OjJMol, or 3OpMol QII2 oxidised per minute per mg protein.

As described in the examples, the oxidation of uhiquinol-l by the rAOX of the invention displayed typical Michaelis-Menten kinetics (Km of 332 xM and I 1 of 30 imo1-'min-1nig-'), a turnover number of 20 jiniol s 1 and remarkable stability. The rAOX was stimulated upon contacting with pyruvate (10mM) and EDT-20 (0.025%) indicating that the recombinant enzyme retains biochemical properties similar to the native protein. Surprisingly, EDT-20 decreased the Km of the rAOX for Q1H2 from 332puM to i01d'd and increased l'mn to 37.8 jmol 1 mm 1 mg1, thereby suggesting that the correct conformational state of the protein is required to achieve maximal activity. -s-

it will he appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substannally the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof. The terms "substantially the amino acid/polynucleotide/polypeptide sequence", "functional variant" and "functional fragment", can be a sequence that has at least 40% sequence identity with the amino acid/polynucleotide/polypeptide sequences of any one of the sequences referred to herein, for example 40% identity with the nucleic acid sequence identified as SEQ II) No:l (i.e. DNA sequence of plant AOX, or 40% identity with the polypeptide identified as SEQ ID No.2 (i.e. protein sequence of plant AOX), and so on.

Amino acid/polvnucleotide/polvpeptide sequences with a sequence identit which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged. Preferably, the amino acid/polynucleoticle/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95°/h identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein.

The skilled technician will appreciate how to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences. In order to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value.

The percentage identity for two sequences may take different values depending on:-(i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score mattix used (e.g. BLOSUM62, PAM25O, Gonnet etc.), and gap-penalty, e.g. functional form and constants.

having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of ecluivalenced positions excluding overhangs. Furthermore, it will he appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.

Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustaIW Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson etal., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way fbr generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity.

For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: LNDGAP = -1, and GAPDIST = 4. Those skilled in the art will he aware that it may he necessary to vary these and other parameters fbr optimal sequence alignment.

Preferably, calculation of percentage identities between two amino acid/polynucleotide/polypeptide sequences may then be calculated from such an alignment as (N/'fltlOO, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps hut excluding overhangs. Hence, a most preferred method for calculating percentage identity between two sequences comprises @) preparing a sequence alignment using the ClustaIW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and 1 into the following formula:-Sequence Identity = ©/T)* 100.

Alternative methods for identifying similar sequences will be known to those skilled in the art.

For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to the sequences shown in SEQ ID No: I, or their complements under stringent condinons. By stringent condinons, we mean the nucleotide hybridises to filter-hound DN A -10 -or RNA in 3x sodium chloride/sodium citrate (SSC) at approximately 45°C followed by at least one wash in 0.2x SSC/0.l°/h S1)S at approximately 20-65°C. Alternatively, a substantially similar polvpeptide may differ hvat least 1, hut less than 5, 10,20,50 or 100 amino acids from the sequence shown in SEQ ID No: 2.

Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could he varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change. Other suitable variants are those having homologous nucleotide sequences hut comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example small non-polar, hydrophobic amino acids include glycine, /5 alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore he appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will known the nucleotide sequences encoding these amino acids.

All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference \vill now be made, by way of example, to the accompanying diagrammatic drawings, in which:-Figure lisa plasmid map of pAOSG/R; Figure 2 is a plasmid map of pETI Sb; -Il -Figure 3 is a schematic representation of one embodiment of an expression construct according to the invention, pET.SgAOX; Figure 4 shows SDS-PAGE analysis of recombinant S. gutta.turn A0X rSgA0X9; Figure 5 shows a Western blot of the SgAOX; Figure 6 shows the oxygen uptake by B. co/i membranes expressing rSgAOX and the effect of the inhibitor, ascofuranone, on the rate of respiration. Rates are expressed as imol 02 consumed/mm/mg protein; and Figure 7 shows the effects of EI)T-20 on the specific activity of the rSgAOX of the invention. /0

Examples

Strains H. coli AhemA mutant (EN 102) strain, which lacks quinol oxidase activity of the cytochrome ho and hd complexes (Nihei et al., 2003, FEBS Left. 538.35-40), was used for the expression i of recombinant alternative oxidase rA0X.

Plasmici construction S. guttatum A0X (SgAOX) lacking a mitochondrial localization signal sequence (SEQ ID No:1) was expressed in H. co/i. The cleavage sites were predicted using MitoProt http://i1ig2.he1rnholtz-muenchen.de/ihg/ntitoprot.htntl; M.G. Clnros, P. Vincens.

Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur. J. Biochem. 241, 779-786 (1996)). In order to remove the leader signal sequence and facilitate cloning, a recognition site for NdeI was introduced at the SgAOX cleavage site. Firstly, the orientation of the SgAOX cI)Ni\ in pAOSGBI (Rhoads, I). M., and McIntosh, L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 21 22-2126) was reversed by digestion with EcoRl, followed by ligation of the resulting fragments to give pAOSG /R, as shown in Figure 1. This plasmid, together with PCR primers SHQ ID No.3 and SRQ ID No. 4 which are shown below), were used to incorporate the NdeI site (alteration underlined), and was performed using the Quick-Change mutagenesis kit (Stratagene.

SEQ ID No. 3 (primer 5'-gttctcgcccccccgccaTATgagcacgctgtcagc-3' -12-SEQ TI) No. 4 (primer): 3'-gctgacagcgtgctcATAtggcgggggcgagaac-3' The mature SgAOX sequence was then removed on a NdeT-BamHT fragment and llgated to NdeT-BamHT digested pETI 5h, which is shown in Figure 2, to ultimately produce tile expression construct pE'f.SgAOX, as shown in Figure 3.

Expression of 3g/IOX in B. co/i membranes L. coll (FNIO2) cells were transformed with the pLT.SgAOX construct shown in Figure 3, and grown overnight on selective Luria agar supplemented with 100p.g/ml amino-levulinic acid (ALA), 50p.g/ml kananwcin and lOOp.g/ml ampicillin. FN102 is a slow grower, and has the wrong complement of en2ymes to make respiratory proteins. This particular strain of E. cob lacks some en7ymes that would normally allow the H. coli to grow. The expression of AOX rescues the organism by allowing it to synthesise a terminal oxidase. Thus, use of this strain is important for the funcrional expression of AOX separate from other terminal oxidases.

A single colony was used to streak a fresh agar plate with the same supplements, and was incubated for 12 hours at 37'C.A scrape of cells from the streak plate was used to inoculate 50m1 starter culture (Luria broth, i00tg/ml ALA, 50p.g/ml kanamycin, 501g/nll ampicillin).

The starter culture was grown at 37CC with shaking fbr -4 hours, centrifuged at B000g fi)r S minutes and re-suspended in 5m1 of non-supplemented Luria broth to remove the ALA from the media. The centrifugation and re-suspension step were repeated, and the resultant cell suspension was used to inoculate SL of S-broth (SOg tryptone-peptone, 25g yeast extract, 25g casamino acid, 32g dipotassium hydrogen orthophosphate, i 5g potassium dibydrogen orthophosphate, 3.7g trisodium citrate, l2.3g ammonium sulphate, 0.25g magnesium sulphate, 0. i 25g iron sulphate, 0.1 25g iron chloride, I DOg glucose and O.5g carhenicillin). The cultures were incubated by shaking at 300C until the 0D600 = 0.1, which usually took about 3.5 hours, at which point the cells were induced with 2SjiM 1soproøyi--[)-thio-galactoside TPTG). After induction, the cultures were incubated for a further 14 hours at 30°C with shaking.

-13 -Following the 14-hour growth period, cells were harvested by centrifugarion at B000g (6 minutes) and cell pellets were re-suspended in SOmMTris-HC1, lOmMpyruvate, pH 7.5.

After tile pellets were pooled and homogenised, a protease inhibitor cocktail (Roche "Complete") was added, before lysis using a French Press (10k psi, two passes). After lysis, cell debris was removed in a single I 2,000g centrifugation step, and the supernatant was centrifuged for 1 hour at 200,000g. The pellets containing the cell membranes were then re-suspended in a minimal volume of 50mM Tris-HC1, 10mM pyruvate, pH 7.5 prior to snap freezing, storage or experimentation.

So/uhi/itation from I i. co/i membranes Membranes were treated with solubili2ation buffer (6 mg/nil protein in 50 mM Tris-l-IC1, 10mM pyruvate, 1% (w/v) n-Dodecvl--D-ma1topvranoside DDM), 10%(v/v) glycerol, pIT 7.5) at 4°C and immediately ultracentrifuged at 200,000 g for 1 hr at 4°C. The uhiquinol oxidase activities of the samples before centrifugation, as well as of supernatant and pellet, were then determined.

Purification of rA OX The hybrid hatch/column procedure described in manufacturer's instruction was used as stated below. 1 ml of the resin (Sigma, His-Select cobalt resin) was equilibrated in a batch format by 3m1 of equilibration buffer (30mM Tris-HC1, I % Qv/v 1)1)M, 100mM Mg504, 10% (v/v glycerol, pu 7.5). 10 ml of DDM extract was mixed with the resin for 1 hour at 4°C. The resin was washed twice with 2.Sml of wash buffer (50 mM Tris-HCJ, 20 mM intidazole, 0.5% DDM, 0.5% (w/v CI2EB (Sigma, 100mM MgSO4, I0%(v/v glycerol pH 7.5) and the resin-bound rAOX was transferred to a column. Finally, rAOX was eluted with increasing imida2ole concentrations by mixing elution buffer (50 mM Tris-HC1, 250 mM imidazole, 0.5% DD1M, 0.5% C12E8, 100 mM MgSO4, l09/o(z/v glycerol p11 7.5 and wash buffer (as above). 1 ml fractions were collected.

Uhiquino/ oxidase assay rSgAOX (i.e. a uhiquinol oxidase oxidizes uhiquinol into uhiquinone and reduces oxygen to water. tJbiquinol oxidase (i.e. rSgAOX activity was measured by recording the change in -14-absorhance of uhiquinol-l at 278 nm (Varian Carv 4000 spectrophotometer. EDT-20 (also known as PEG-1(3 Tallow aminopropylamine, or N-(Tallowalkyl)trirnethylenediamine, obtained from Sigmas Chemical Co. was added immediately to a final concentration of 0025% (v/t), prior to the addition of the sample containing the rSgAOX. Reactions were started by the addition of rSgAOX-containing sample after the addition of uhiquinol-l (final concentration 150 jaM, 8275 = 15,000 M-1cnv1) to a reaction buffer containing 50 mM Tris-HC1 (ph 7.5) and 10mM pyruvate.

Oxygen uptake Respiratory activity of the rSgAOX was measured with a Clark-type electrode (Rank Brothers, Cambridge, U.T<.) using 0.1 to 0.5 mg Ii. cole membranes suspended in 0.4 mL air-saturated reaction medium (250 jaT\1 at 25 °C, R.R, Wise, AW, Naylor, Calibration and use cIa clark-type oxygn-elcctrodc from S to 45 °C, Anal. Biochem. 146 (1985) 260-264) containing 50 mM Tris-HCI (pH 7.5).

W'7esten analysis Separation of mitochondrial proteins on reducing (5mM Dithiothreitol (1)11)) 51)5-polyacrylamide gels, followed by their transfer to nitrocellulose membranes and the detection of AOX protein using monoclonal antibodies raised against the S. guttaturn AOX was performed as described previously (M.S. Aibury, C. Allourtir. A.T. Moore, A highly conserved glutamate residue (L270) is essential ibr alternative oxidase activiry, J, Biol. Chem. 273 (1998)30301 --30303.

Càlletochlorin B synthesis Colletochlorin B was synthesised using the technique described by K.-M. Chen and M. M. Joullie (Tetrahedron letters, 23, 4567-4568, 1982 Synthesis of Colletocblorin B), using geranyl bromide as the alkylating agent in the final step, resulting in a white product (20%).

General Molecular Biology Procedures Ollgonucleotides were obtained from MWG Biotech. Sequencing was performed by Beckman Coulter Genomics. Other procedures were as described by Samhrook c/ aU.

Sambrook, Fritsch, 1* 17., and Maniatis, TI., Molecular Cloning: A Laboratory Manual, Cold -15 -Spring I larhor Laboratory, Cold Spring harbor, NY, 1989. All chemicals were biochemistry grade. tJbiquinone-1 was purchased from Sigma-Aldrich, and the protease inhibitor cocktail' was obtained from Roche.

Example 1 -Purification of full active recombinant Scmrornaiwn uu//a/wn AOX (rSgAOX) Although the inventors previously established a protocol for the functional expression of AOX from Anim macn/a/urn spadices, it was dependent upon the seasonal availability of spadices, and furthermore, the yield of the active en7yme was too low for crystallographic studies (Affourtit, C. and Moore, A.L. (2O04 Biochim Biophys Acta 1608, 181-189 "Purification of the plant alternative oxidase from ztinim macna/sm?: measurement, stability and activaflon"). Therefore, the conditions for the high level expression of recombinant Sa.uromatsmi ga/ta/urn AOX rSgAOX, and purification protocols were significantly optitni'i.ed in order to obtain large quantities of active and stable rSgAOX. The inventors found that several factors were believed to be important for obtaining large amounts of active rSgAi) X. Importantly, the presence of pyruvate was believed to important as it causes a conformational change in the rSgAOX, which, as discussed below, caused a surprising increase in the overall activity of the en7yme. Furthermore, the pyruvate also stahilises the enzyme by some unknown mechanism. In addition, the inclusion of EDT-20, as it stabilises the en2yme in its active-form, possibly by mimicking the mitochondrial membrane. Thus, the presence of pyruvate and FA)T-20 were believed to be important hut for somewhat different reasons.

Other features of the method which improved the isolation of rSgAOX were the growth time of the k. co/i culture expressing the enzyme prior to addition of the inducer compound Isopropyl--D-thio-galactoside (IPIG), and the concentration of the inducer.

After extensive screening of detergents and additives to establish the procedure for efficient extraction of active rSgAOX from die inner membranes of die host B. co/i cells, die inventors found that 1% (n;/v n-dodecyl-jJ-D-maltopyranoside (DDM) specifically soluhilized rSgAOK

as shown in Table 1. -16-

Table 1 -Purification of rSAOX Fractions Total activity Protein Specific activity Recovery Eumol/min) (mg imol/min/mg (%) Inner membrrne 29.7 32.5 0.91 100 1)DM extract 29.5 31.5 0.93 99.3 Co-column 14.6 0.71 20.6 49.1 The activities listed in Table I were measured by using 150 aM of ubiquinol-l. Inner membrane was prepared from I litre cultures, and fractions were collected as purified rSgAOX after Co-column. It should be noted that this purification was performed in the absence of 10mM pyruvate.

Approximately 100% of the membrane quinol oxidase rSgAOX) activity was recovered with 1 Yo (w/v D1)M in the extract. Thus, the recovery of the activity demonstrated here was /0 significantly higher than the 17% activity that was previously reported using the BigGI lAP extraction technique (Affourtit, C. and Moore, AL (2004) Biochim l3iophys Acta 1608, 181- 189 "Purification of the plant alternative oxidase from Anini maculatum: measurement, stability rnd activation"). Following solubilization, it was possible to maintain en7ymatic activity for at least one month at 20°C.

H

Since rSgAOX was fused with N-terminal histidine tag, soluhilized rSgAOX was purified by cobalt affinity chromatography. The enzyme soluhilized by 1)1)M was bound to the cobalt affinity resin in the presence of DDM, but in contrast to recombinant trvpanosome alternative oxidase rTAO), rSgAOX bound to the resin could he eluted with buffer containing 0DM. Finally, purified rSgAOX was obtained by elution with 250 mM imidazole resulting in a very efficient purification of active rSgAOX.

Example 2-Characterisation of rSirAOK The purified rSgAOX, with a molecular mass of 34 kDa, was estimated to be at least 97% pure by SDS-PAGE, as shown in lane 4 of Figure 4. In addition, it is apparent that other bands could also he identified including two bands with a smaller size than rSgAOX and one -17 -hand with an approximate molecular mass of 74 Since all of these hands were recogni7ed in the Western blot using a monoclonal anfihody against SgAOX (see Figure 5), the smaller protein bands possthly represent proteolytic breakdown products whilst the 74 lcDa band represents a dimer of rSgAOX.

The specific activity of the purified rSgAOX was found to he more than 30 mol-'min-'mg-' when 150 jiM of uhiquinol-l was used substrate in the presence of I0mv1 pyruvate.

Quinol oxidase activity, of purified rSgAOX, as measured by the oxygen electrode (see Figure 6), was insensitive to I mM KCN or luM antinwcin A, but was completely inhibited by 10 /0 ni'vl ascofuranone, as shown in Figure 6. It should he noted that Figure 6 shows the rate in pmol 02 consumed/min/ntg protein, which is equivalent to 30 pntol ubiquinol oxidised/min/mg protein. As summarized in Table 1, a greater than 22-fold increase in purification was achieved using the techniques described above, and 49.1 Yo of the total activity was recovered from the lysate of FN1O2/pSgAOX cells. Such procedures resulted in i approximately 5mg of highly purified rSgAOX from a 5/culture.

1 able 2 indicates the sensitivity of the purified rSgAOX protein to a range of AOX inhibitors including SHAIvI, n-propyl gallate, ascofuranone and colletochlorine B. It is readily apparent from the TCSI) values that both ascofuranone and its derivative, colletochlorine B, are much more potent than the other AOX inhibitors with IC50 values ranging from 5-2OnM.

1 able 2 -Sensitivity of the purified rSgAOX protein to a range of AOX inhibitors Inhibitor 1C50 Colletocli] arm B 1 O5pM Ascafurananc 5SpM Octvl Gallate lO5nM Salicvlliydraxaniic acid IIDQ 24OnM -18 -Kinetic analysis of purified plant SgAOX using uhiquinol analogues has previously proved to he difficult because the natural substrate of the plant AOX is ubiquinol-1 0 (C. Affourtit et al., 1999.J. Biol. Chem. 274: 6212-6218; M.H.N. Hoefnagel etal., 1998, Arch Biochem & Biophys 355: 262-Affourtit, C. and Moore, A.L. (2004) Biochim Biophys Acta 1608, 181-1 89 "Purification of the plant alternative oxidase from Arwn macu/a/mn: measurement, stability and activation"), \vhich is too hydrophobic to use as the substrate in the assay, and the enzymatic activity was not saturated at the maximum concentration of duroquinol (approximately 300 ulYl). However, since the inventors have purified tSgAOX in its fully active form, the purified enzyme was very well-suited for kinetic analysis.

The linear relationship between the substrate concentration and the rate of oxidation of ubiquinol-1 has also previously been observed in both Trypanosome Alternative Oxidase (TAO) and Arum j/a/jcmm AOX (M.II.N. lloefnagel et al., 1998, Arch Biochem & Biophys 355: 262-270, and Hoefnagel etai., 1998 reported that the addition of a specific detergent (0.025Vo EIJT-20) during the assay increased the activity 3-to 4-fold dose to saturation.

Referring to Figure 7, there is shown the effect of adding EI)T-20 to the activity of the rSgAOX. It is apparent from Figure 7 and fable 3 that the addition of 0.025(w/t) °A of EDT-also significantly enhanced the activity of purified rSgAOX approximately 2-fold decreasing the Km for Q1H2 from 332fv[ to 10liM and increasing the Vm,x from 30.2 1imoF' mm-' mg-1 to 37.8 iimol-' min' mg1.

Table 3-Summary of rSgAOX kinetics in the presence and absence of E1)T-20 w/ ED'f-20 xv/o EDT-20 Km (pM 101 332 Vrnax (jJmol/rnin/rng 37.8 30.2 kcat = 22.5 /sec kcai/Km = 0.22 pmol sec -19 -

Summary

Tn summary, the inventors have devised a novel method for the over-expression of recombinant SgAOX (rSgAOX, in a Ahemr4-deficient Eschcrichia co/i strain (FNIO2). As this strain of P. co/i lacks some enzimes that would normally allow the P. co/i to grow, the expression of AOX rescues die organism by allowing it to synthesise a terminal oxidase, thereby enabling the functional expression of AOX separate from other terminal oxidases.

B. co/i membranes were solubilized and purified to homogeneity in a very stable and active form. SDS gels and Western blots revealed a doublet band located at approximately 32kDa.

The oxidation of uhiquinol-1, by purified rSgAOX, displayed typical Michaelis-Menten kinetics (K1 of 332 uM and 1/ of 30 jLmol 1min mg1), a turnover number of 20 tmol s 1 and remarkable stability. The purified recombinant protein was stimulated by 10mM pyruvate (which is a keto acid) and 0.0257o FDT2O similar to that observed with the protein isolated from thermogenic plants indicating that the recombinant protein retains biochemical 1 properties similar to the native protein. The pyruvate is believed to cause a contormational change in the rSgAOX, which results in a surprising increase in the overall activity of the enzyme. The pyruvate also stabilises the enzyme. The EDT-20 stabilises the enzyme in its active-form, possibly by mimicking the mitochondrial membrane. Of particular interest was the finding that FJ)T-20 decreased the T<m for Q1H2 from 3321fv1 to 1 Ol1iM and increased Turn to 37.8 mol min mg1, suggesting that die correct conformational state of the protein is required to achieve maximal activity. rSgAOX was potently inhibited not only by conventional inhibitors, such as SHAM and n-propylga]iate, but also by the potent TAO inhibitors ascofuranone and an ascofuranone-derivative colletochlorin B. It is anticipated that highly purified and active AOX will open new directions with respect to the investigation of the structure and reaction mechanisms of AOXs through the provision of large amounts of purified protein fur crystallography and contribute to further progress of the rational design of phytopathogenic compounds.

Claims (1)

1. <claim-text>-20 -Claims 1. A method of preparing recombinant Alternative Oxidase rAOX9, the method comprising culturing a host cell comprising a genetic construct comprising a nucleic acid molecule which encodes an alternative oxidase (AOX) under conditions suitable for the production of rAOX; and contacting the rAOX with a keto acid.</claim-text> <claim-text>2. A method according to claim 1, wherein the lceto acid alpha-lceto acid, a beta-keto acid or a gamma-keto acid.</claim-text> <claim-text>3. A method according to either claim 1 or claim 2, wherein the keto acid is an alpha-keto acid.</claim-text> <claim-text>4. A method according to any preceding claim, wherein the keto acid is pyruvic acid (i.e. pvruvate, oxaloacetic acid or glycolic acid. /5</claim-text> <claim-text>3. A method according to any preceding claim, wherein the keto acid is pyruvate.</claim-text> <claim-text>6. A method according to any preceding claim, wherein the concentration of keto acid contacted with the rAOX is at least 1mM, 2mM, 3mM or 8mM.</claim-text> <claim-text>7. A method according to any preceding claim, wherein the concentration of keto acid is between lmtvl and 10mM, or between 2mv1 and 10mM, or between 3miM and l0mvl.</claim-text> <claim-text>8. A method according to any preceding claim, wherein the keto acid forms part of a buffered medium, for example Tris-HCI.</claim-text> <claim-text>9. A method according to claim 8, wherein the pIT of the buffered medium is between 7.0 and 8.0.</claim-text> <claim-text>10. A method according to any preceding claim, wherein the method comprises contacting the rAOX with an emulsifying agent.</claim-text> <claim-text>-21 - 11. A method according to claim 10, wherein the emulsifying agent is LDT-20.</claim-text> <claim-text>12. A method according to either claim 10 (Jr claim 11, wherein the concentration of the emulsifying agent is at least 0.01% v/v, 0.02% v/v or at least 0.025% v/v.</claim-text> <claim-text>13. A method according to any one of claims 10 to 12, wherein the concentration of emulsifying agent is between 0.01?/ and 0.025?/ (v/v), or between 0.02% and 0.025% (v/v).</claim-text> <claim-text>14. A method according to any preceding claim, wherein the host cell is a yeast cell, a /0 fungal cell or a bacterial cell.</claim-text> <claim-text>15. A method according to any preceding claim, wherein the host cell is a haem-deficient strain.</claim-text> <claim-text>/5 16. A method according to any preceding claim, wherein the host cell is B. co/i ShemA mutant (FNI 02) strain, which lacks quinol oxidase activity of the cytochrome ho and hd complexes.</claim-text> <claim-text>17. A method according to any preceding claim, wherein the AOX is a plant AOX, a fungal AOX or a bacterial AOX.</claim-text> <claim-text>18. A method according to any preceding claim, wherein the nucleic acid molecule, which encodes the rAOX, comprises a nucleotide sequence substantially as set out in SEQ ID No:1, or a functional variant or a fragment thereof.</claim-text> <claim-text>19. A method according to any preceding claim, wherein the rAOX comprises an amino acid sequence substantially as set out in SEQ ID No: 2, (Jr a functional variant (Jr fragment thereof.</claim-text> <claim-text>20. A genetic construct substantially as represented in Figure 3.</claim-text> <claim-text>-22 - 21. A construct according to claim 20, wherein the construct comprises a nucleotide sequence substantially as set out in SEQ Ti) No: 1, or a functional variant or a fragment thereof and/or encodes a rAOX, which comprises an amino acid sequence substantially as set out in SEQ ID No: 2, or a functional variant or fragment thereof.</claim-text> <claim-text>22. Recombinant Alternative Oxidase rAOX obtained by, or obtainable from, the method according to any one of claims 1 to 19.</claim-text> <claim-text>23. The rAOX according to claim 22, which is stable for at least 1, 2, 3, 4, 5 or 6 months. /0</claim-text> <claim-text>24. The rAOX according to either claim 22 or claim 23, wherein the activity of the rAOX is greater than IOpMol, 2OpMol, or 3OjJMol Qil? oxidised per minute per ntg protein.</claim-text>