WO2010015264A1 - L-asparaginase from helicobacter pylori - Google Patents

Abstract

The present invention discloses a new L-asparaginase from Helicobacter pylori. The recombinant enzyme cloned from Escherichia coli has been isolated, purified and characterized. It can be used as a new therapeutic and diagnostic tool. Further, its application as a food additive has been disclosed.

Description

DESCRIPTION

L-ASPARAGINASE FROM HELICOBACTER PYLORI The present invention concerns the recombinant form of L-asparaginase from Helicobacter pylori CCUG 17874; in particular, the relevant gene has been cloned, the corresponding enzyme expressed and then characterised. Antibodies against the L-asparaginase of the invention have been produced. In addition, the use of said enzyme as a potential therapeutic agent, as a reagent in a diagnostic assay and as a food additive is also disclosed.

BACKGROUND OF THE INVENTION

L-asparaginases (EC 3.5.1.1) are amidohydrolases that primarily catalyse the conversion of L-asparagine to L-aspartate and ammonia. A minority of L-asparaginases, also referred to as glutaminases-asparaginases (EC 3.5.1.38), are enzymes capable of transforming either L- asparagine or L- glutamine into their corresponding acids with comparable efficiency. L- asparaginases play a central role in the metabolism of several species, the aspartate being either transaminated to oxaloacetate, an intermediate in the tricarboxylic acid cycle, or converted into fumarate during the urea cycle. L-asparaginases are present in bacteria, plants, animal tissues and in the serum of certain rodents (El-Bessoumy et a/., 2004), and an asparaginase-like protein of human spermatozoa has been recently reported (Bush et a/., 2002). Bacterial L-asparaginases which are preferentially active towards L-asparagine are in turn subdivided into two groups according to their affinity towards L-asparagine and to their cellular localisation. Type I L-asparaginases are cytoplasmic, display high Km values vs. L-asparagine and are also active towards L-glutamine. Type Il L-asparaginases are periplasmic, exhibit low Km values vs. L-asparagine, and have low-to-negligible activity towards L-glutamine. Bacterial L- asparaginases are known to exhibit a hyperbolic response to L-asparagine (Mickalska and Jaskolski, 2006; Sanches et a/., 2007). Nevertheless, it has been recently reported that L-asparaginase I from Escherichia coli (E. coli) displays positive cooperativity towards L-asparagine and is allosterically regulated by the substrate itself (Yun et al., 2007). The three-dimensional structures of L-asparaginase from different bacterial sources have been extensively studied (Lubkowski et al., 1996; Miller et al., 1993; Swain et al., 1993; Yao et al., 2005; Yun et al., 2007). In their highly conserved architecture, they are 140-150 kDa homotetramers built up by identical subunits of 300 to 350 amino acid residues. The tetrameric structure can be more accurately described as a dimer of intimate dimers (Swain et al., 1993), with each subunit consisting of two alf a/beta domains, a larger N-terminal domain and a smaller C-terminal domain, connected by a structured linker region. Two catalytic site pockets lie at the intersubunit interface of the intimate dimer, each formed by residues that are asymmetrically donated by both subunits. Therefore, four independent catalytic sites are present in the homotetramer, each created by a rigid part located in the protein core and by a highly flexible N- terminal loop largely exposed to the solvent and forming a lid over the active site (Lubkowski ef al., 1996; Swain ef al., 1993). Several structural and kinetic studies have suggested that the catalytic reaction of L- asparaginases proceeds through the classic two-step ping-pong mechanism observed in serine proteases (Carter and Wells, 1988). Actually, the enzymatic activity is likely to depend on two sets of highly conserved amino acid residues, known as catalytic triads and represented by Thr12-Tyr25-Glu283 and Thr89-Asp90-Lys162, respectively, in E. coli L-asparaginase (Dodson and Wlodawer, 1998; Sanches ef al., 2007). Some L-asparaginases from bacterial sources display anti-leukemic activity, and type Il E. coli L-asparaginase and the related enzyme from Erwinia chrysanthemi (E. chrysanthemi) are currently in clinical use as effective drugs in the treatment of acute lymphoblastic leukemia (ALL) and other lymphoid malignancies. The anti-tumour activity is primarily attributed to the reduction of L-asparagine in blood. In fact, the depletion of L-asparagine selectively inhibits the proliferation of malignant cells that, in contrast to normal cells, are dependent on an exogenous source of this amino acid for survival because of a decreased or absent asparagine synthetase activity (Keating et al., 1993; Moola et al., 1994). Although the anticancer properties were demonstrated for different bacterial asparaginases (Boyse et al., 1967; Cammack et al., 1972; Ehrman et al., 1971 ; Kotzia and Labrou, 2007; Krasotkina et al., 2004; Wriston and YeINn, 1973), their practical applications are highly restricted by the side effects associated with the therapy, i.e., hypersensitivity reactions, L- asparaginase resistance, liver damage, acute pancreatitis and other disturbances. It is thought that a number of the medical drawbacks are linked to the associated glutaminase activity, which leads to depletion of the circulating L-glutamine (Reinert et al., 2006; Distasio et al., 1982, Ollenschlager et al., 1988; Gallagher et al., 1989). Therefore, novel L- asparaginases are more and more investigated in order to identify more effective enzymes with less toxic effects (e.g., higher Km values versus L- glutamine, longer circulation times, lack of immunological cross-reactivity, etc.).

In addition, asparagine is known to react with reducing sugars contained in starch-based foods during processing like baking of frying or heating above 120° C and is thus responsible for the production of acrylamide as a product of a Maillard reaction (see, for instance, ZyzakD. , J. Agric. Food Chem. 2003, 51 , 4782-4787 or Mottram et al. Nature, 419:448, 2002). Concerns about dietary exposure to acrylamide had arisen as a result of studies conducted in Sweden in 2002, which showed that high levels of acrylamide were formed during the frying or baking of a variety of foods. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) reviewed the safety of acrylamide in 2005 and recommended that acrylamide be re-evaluated when results of ongoing carcinogenicity and long term neurotoxicity studies, which are being conducted around the world, become available and that appropriate efforts to reduce acrylamide concentrations in food should continue.

Thus, there is a strong need for L-asparaginases, which would be more stable and resistant to pH changes and high temperature, possibly being also more selective toward asparagine than glutamine, thus leading to less side effects and allowing to be used for improved therapeutic and diagnostic purposes or even as a food additive. In fact, there is a quest for new antigens to be used as reagents to diagnose patients with a suspect of ongoing or previous infections sustained by Helicobacter pylori, while anti-asparaginase antibodies might be useful in research investigations focused on the pathogenicity role of this enzyme.

SUMMARY OF THE INVENTION It has been surprisingly found that L-asparaginase from Helicobacter pylori CCUG 17874 is particularly advantageous with respect to other known asparaginases. In fact, it has unexpectedly shown a significant specificity toward asparagine rather than versus glutamine, with respect to other known asparaginase. Again, L-asparaginase from Helicobacter pylori CCUG 17874 proved to be more stable both to wide pH changes and to high temperature.

The gene encoding the L-asparaginase from H. pylori CCUG 17874 of the present invention has been cloned and sequenced. Further, a vector comprising said gene has been transformed into E. coli in order to produce suitable quantities of the recombinant enzyme, which has then been purified and characterized.

In addition, monoclonal antibodies against the recombinant L- asparaginase from Helicobacter pylori CCUG 17874 have been produced and purified. The recombinant protein has also been used for developing an immunological diagnostic assays, whose preliminary data show that it can be more sensitive compared to the commercially available diagnostic assays in detecting patients affected by Helicobacter pylori. The recombinant L-asparaginase from Helicobacter pylori CCUG 17874 of the invention may advantageously be used also as a food additive in order to convert L-asparagine present in starch-based foods to L-aspartate and ammonia, thus reducing the amount of acrylamide formed during food processing. OBJECT OF THE INVENTION

It is therefore a first object of the invention a sequence of a nucleic acid encoding the L-asparaginase of Helicobacter pylori CCUG 17874.

In particular, said nucleic acid sequence is a deoxyribonucleic acid

(referred to also as DNA) sequence corresponding to Seq. ID n. 1.

In addition, said DNA sequence is the one coding for the amino acid sequence of Seq. ID n. 2. A second object is represented by a transformation vector comprising the nucleotide sequence of Seq. ID n. 1.

A third object of the invention, therefore, is represented by an expression host comprising the transformation vector of the invention.

A fourth object of the invention is represented by a method for producing recombinant L-asparaginase from Helicobacter pylori CCUG 17874.

In an additional embodiment, the present invention also concerns a method for the preparation of antibodies against the recombinant L- asparaginase of the invention.

It is a further object of the present invention to provide antibodies specific for the L-asparaginase from Helicobacter pylori CCUG 17874.

A method for determining the presence in a sample of the antibodies against the recombinant L-asparaginase from Helicobacter pylori CCUG

17874 represents a still further embodiment.

A diagnostic kit for determining the presence in a sample of the antibodies against the recombinant L-asparaginase from Helicobacter pylori CCUG

17874 is another embodiment of the invention.

A further additional embodiment of the invention is represented by the use of the recombinant L-asparaginase from Helicobacter pylori CCUG 17874 as a medicament and, in particular, as an anti-cancer medicament. A pharmaceutical preparation comprising the recombinant L-asparaginase from Helicobacter pylori CCUG 17874 of the invention is a still additional embodiment of the present invention. The use of said pharmaceutical preparation for the treatment of cancer, acute lymphatic leukemia (ALL) and other diseases is comprised within the present invention as well.

In addition, the use of the recombinant L-asparaginase from Helicobacter pylori CCUG 17874 of the invention as a food additive and a method for reducing the production of acrylamide during food processing represent a further object of the present invention.

DETAILED DESCRIPTION OF THE INVENTION According to the first object, the present invention encompasses the nucleic acid molecule of Seq. ID n. 1 encoding L-asparaginase from H. pylori CCUG 17874 having the amino acidic sequence of Seq. ID n. 2. In particular, said nucleotide sequences may be a sequence of deoxyribonucleic acid (DNA), complementary DNA (cDNA), ribonucleic acid (RNA), ESTs, chromosomes or genes, both sense and anti-sense, single-stranded or double-stranded or, where applicable, their complementary sequences using the Watson-Crick base pairing, or any sequence hybridizing under stringent conditions, i.e. low ionic strength and high temperature for washing, as well as any fragments thereof. In a preferred embodiment, the nucleic acid molecule is a DNA sequence coding for the amino acidic sequence of Seq. ID n. 2. In addition, there are also included the sequences, which, due to the degeneracy of the genetic code, encode the same amino acidic sequence of Seq. ID n. 2, that is to say that more than one nucleotide sequence may encode the same amino acidic sequence of Seq. ID n . 2 as a result of one or more silent mutations.

Moreover, the present invention also encompasses homologous sequences to that of Seq. ID n. 1. "Homology degree" between two nucleotide sequences is defined as the percentage of nucleotides occupying the same position when the sequences are aligned. Alignment may be performed according to various algorithms or programs, such as, for instance, FASTA, BLAST, BLOUSM62 or ENTREZ. An homology of 100% means that the sequences are identical. Preferably, the nucleotide sequences of the invention are identical to the nucleotide sequence of Seq. ID n. 1. Alternatively, the nucleotide sequences of the invention have a variable homology to the sequence of Seq. ID n.1. Within the scope of the present invention, there are also included homologous sequences to those of Seq. ID n. 2, wherein the degree of homology as defined above applies as well. Preferably, the amino acidic sequences of the invention are identical to the amino acidic sequence of Seq. ID n. 2. Alternatively, the amino acidic sequences of the invention have a variable homology to the sequence of Seq. ID n. 2.

In addition, there are also encompassed analogous, and more in particular, non-natural analogous amino acidic sequences to that of Seq. ID n. 2, wherein "analogy" is defined as the percentage of structurally related amino acids occupying the same position. "Structurally related" amino acids are isosteric and/or isoelectric amino acids, i.e. those belonging to the same of the following groups: basic amino acids, such as: lysine, histidine and arginine, acidic amino acids, such as: aspartic acid, glutamic acid and their amide derivatives asparagine and glutamine, non- polar amino acids, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, metionine, tryptophan and uncharged amino acids, such as glycine, asparagine, glutamine, cysteine, serine, threonine and tyrosine. Therefore, the present invention also encompasses the nucleotidic sequences due to both natural and non-natural mutations, leading to a mutant enzyme, wherein natural mutations are spontaneous events and artificial mutations are introduced. A mutant enzyme is an enzyme that has been produced by a mutant organism, i.e. one which is expressing a mutant gene. A mutant gene (other than one containing only silent mutations) means a gene encoding an enzyme having an amino acid sequence which has been derived directly or indirectly, and which is different in one or more locations from the sequence of a corresponding parent enzyme, being the product of the corresponding unaltered gene. On the contrary, a silent mutation in a gene results in the same amino acid sequence of the enzyme being encoded by that gene. Beyond the natural events, mutations may be introduced in order to create novel enzymes having novel and possibly better and more useful features. Random mutagenesis is a widespread tool used in molecular biology to create mutated enzymes. For instance, an enzyme with increased activity toward its substrate or higher specificity for a substrate with respect to another one, may result from mutagenesis experiments.

Among the various well-known techniques, there is the error prone PCR, which is a random mutagenesis technique for introducing amino acid substitutions in proteins. In practice, mutations are deliberately produced into a gene during PCR through the use of error prone DNA polymerases, whose rate of error, together with the number of duplications, defines the mutation frequency, and/or by modifying some reaction conditions, such as temperature, buffer and magnesium ions concentration. The mutated PCR products are then cloned into an expression vector and the resulting library of mutant enzymes is screened for defined changes in the protein activity.

As per the second object, the present invention concerns an expression vector comprising the nucleotide sequences of Seq. ID n. 1 or any nucleotide sequences as per the first object of the present invention. Suitable vectors may be the plasmids, which are circular and double- stranded extra-chromosomal DNA molecules separate from the chromosomal DNA and capable of replicating independently of the chromosomal DNA. Other suitable vectors may be, for instance, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes or yeast artificial chromosomes. Preferably, for the purposes of the present invention, the expression vector is a plasmid and, more preferably, it is the pCR2.1-TOPO cloning vector (Invitrogen) or pET101 , which have been used for cloning and for subcloning, respectively. In addition to the nucleotide sequence of Seq. ID n. 1 of the invention, an expression vector may comprise other sequences, which, according to the type of expression vector used, may be a control sequence, a termination sequence, a promoter sequence, a ribosome binding sequence, all of which are operably linked to the sequence encoding the L-asparaginase of the invention, that is to say that they are into functional relationship with each other. Alternatively, the relevant nucleic acid molecule may be also integrated into the host chromosome. An expression vector is then transferred into a suitable host for expressing the product it encodes.

Transfection and transformation may be carried out according to well known methods in the art, such as, for instance, heat-shock, electroporation, lipofectamine or with the use of bacteriophages or by calcium phosphate (F. L. Graham and A. J. van der Eb). A host comprising the nucleotide sequence of Seq. ID n. 1 , therefore, represents a third object of the invention. Suitable expression host may be procariotic cells, such as Escherichia coli, bacilli such as Bacillus subtilis, Enterobacteriaceae, such as Salmonella typhimurium or Serratia marcesans or various Pseudomonas spp. Yeast cell may be used as well and may be the common bakery-yeast Saccharomyces cerevisiae or Pichia pastoris.

According to the preferred embodiment of the invention, the expression host is a procariotic cell and more preferably it is Escherichia coli BL21(DE3). According to a fourth object, the present invention also relates to antibodies against the L-asparaginase from H. pylori CCUG 17874 of the invention.

Within the meaning of the present invention, antibodies include polyclonal or monoclonal antibodies, i.e. antibodies obtained from a population of substantially homogeneous antibodies directed against a single determinant on the antigen, and they may be monoclonal antibodies of any types, such as, for instance, IgG, IgA, IgD, IgM and IgE, as well as any fragments thereof, such as Fab fragments, F(ab')₂, Fab' and single chain antibodies (scFv).

In a preferred embodiment of the invention, the antibodies are monoclonal IgM antibodies. It is a further object of the present invention a process for the preparation of the recombinant IgM monoclonal antibodies of the invention. Said process includes the step of immunizing mice with a suitable amount of the L-asparaginase of the invention, i.e. an amount capable of eliciting an immune response and, hence, the production of antibodies against said antigen; suitable amounts may be, for instance, from 10 to 100 μg of recombinant H. pylori CCUG 17874 L-asparaginase. Immunization is performed according to methods known in the field of the invention and may include one or more administrations (boosts) of the antigen to the mice, which administrations are performed after some times one from each other; for instance, a first immunization may be followed by the second one after some weeks, such as three or four weeks. As from the Experimental Section, immunization may be made with a starting boost of 30 μg followed by two boosts of 20 μg each 4 weeks from the previous one. The skilled person in the art will be able to optimize the immunization protocol in order to enhance the quantity of antibodies which may be harvested.

Immunized mice are then sacrificed and their spleens harvested in order to recover B cells, which are further fused for immortalization with myeloma cells, such as, for instance, with NSO mouse myeloma cells. Antibodies producing cells are then selected with an ELISA assay using the recombinant L-asparaginase of the invention as a coating antigen. In a further step, the selected clones are expanded for allowing the production of the antibodies, which are separated from the cells by a centrifugation and chromatographic step, for instance, by using a Protein A Sepharose column. In a further embodiment, the present invention discloses a method for the detection in a sample of the presence of IgM and IgG antibodies against the L-asparaginase from Helicobacter pylori CCUG 17874 of the invention. In a preferred embodiment, said method may be an immunologic assay, such as ELISA.

The method, therefore, includes the step of coating an ELISA plate with the L-asparaginase of the invention, which is the antigen. In the case wherein the assay is performed on a serum sample, i.e. the liquid fraction that can be separated from clotted blood, then the above assay may be usefully carried out as a diagnostic tool to detect whether in a patient's serum sample there are present specific antibodies, in particular, IgG and/or IgM antibodies against the L-asparaginase from Helicobacter pylori CCUG 17874. In fact, the detection of IgM antibodies in patients' serum can easily be performed replacing a peroxidase conjugated anti-human IgG antibody with a peroxidase conjugated anti-human IgM antibody.

The McNemar's test, which is a statistical tool well known in the biosciences for the analysis of experimental results, has been used for the interpretation of the results obtained both when a commercial diagnostic method (Diamedix) and a method according to the present invention using the L-asparaginase from Helicobacter pylori CCUG 17874 have been performed. It came out that the proportion of positives detected by the L- asparaginase of the invention used as a single antigen is similar to that detected by the Diamedix kit. Moreover, the L-asparaginase from Helicobacter pylori CCUG 17874 might allow the identification of a 25% portion of the patients negative at the Diamedix kit, indicating that this enzyme might provide an antigen for an improved kit for detecting the patients who have come in contact with the bacterium and that would nevertheless be classified as false negatives according to the commercial kits which typically detect the Cag and Vac antigens. Accordingly, a diagnostic kit comprising the L-asparaginase from Helicobacter pylori CCUG 17874 immobilised onto a plate, represents a further object of the invention.

As per a further additional object of the invention, L-asparaginase may also be used as a medicament for the treatment of cancer.

In particular, acute lymphatic leukemia (ALL) may represent an important target disease, while other ones may be malignant hematologic diseases such as other lymphoma types, leukemia, or myeloma, or non-malignant diseases such as autoimmune diseases (rheumatoid arthritis, Systemic Lupus Erythematosus), and even AIDS.

The pharmaceutical preparation comprising the L-asparaginase from

Helicobacter pylori CCUG 17874 represents another object of the present invention.

Said preparation also includes suitable stabilizers, preservatives and other pharmaceutically and physiologically acceptable eccipients as well known to the skilled person in the art.

The use of said pharmaceutical preparation for the treatment of cancer is encompassed as well within the scope of the present invention. As an additional object of the invention, the L-asparaginase from Helicobacter pylori CCUG 17874 can be used as a food additive in order to reduce the amount of acrylamide formed from the reaction between asparagine and reducing sugars contained in foods and, especially, in starch-based foods. Starch-based foods include, for instance, potato chips, French fries, potato crisps or croquettes, cereals such as rye, corn, maize, barley, rice or oats containing products or wheat based products, which are largely consumed.

Other food products may be represented by coffee, cocoa, nuts, vegetables such as, for example, asparagus or fruits such as, for instance, bananas.

Accordingly, a method for reducing the production of acrylamide due to food processing, and especially during the processing of starch-based foods, comprises the steps of applying the L-asparaginase from

Helicobacter pylori CCUG 17874 of the invention to foods before processing.

In particular, foods may be those listed before, while processing includes, for instance, baking, frying, boiling or roasting or any other processing which is carried out at 120⁰C or even above.

More in detail, it is preferred that the enzyme is applied to foods, for instance, by contacting the enzyme with foods or through the immersion of the foods in a solution containing the L-asparaginase for some times before processing, i.e. before frying, roasting, boiling or heating, in order to allow the L-asparaginase of the invention to act and reduce the quantity of asparagine in food.

According to the advantageous stability properties of the L-asparaginase of the invention, the time required to inactivate the acrylamide producing amino acid, i.e. asparagine, can be reduced compared to the other known enzymes.

DRAWINGS

Fig. 1 Represents the amino acid sequence alignment of L-asparaginase from different strains of H. pylori: from top to bottom: strain CCUG 17874, J99, HPAG1 and HP26695. Dashes indicate the highly conserved regions, dots and colons the sequence differences. The asterisks (*) indicate the first catalytic triad, the § symbol indicates the second catalytic triad (Sanches et al., 2007). Both of them are absolutely conserved throughout the strains. The figure was prepared with Clustalw.

Fig. 2 Shows the amino acid sequence alignments of L-asparaginases from different bacterial sources. The sequence of H. pylori CCUG 17874 L-asparaginase (top) is aligned with the homologous proteins from different species whose structures are deposited in the PDB database. From top to bottom: W. succinogenes (PDB code: 1WSA); E. coli (PDB code: 1 NNS); Pseudomonas 7A (PDB code: 1DJP); A. glutaminasificans (PDB code: 1AGX); E. chrysanthemi (PDB code: 1O7J). The residues highly conserved throughout the different structures are indicated by dashes; by dots and colons or nothing the non-conserved residues. The asterisks (*) indicate the first catalytic triad; the § the second catalytic triad (Sanches et at., 2007). Both of them are absolutely conserved throughout the different species. The figure was prepared with Clustalw.

Fig. 3 Shows the SDS-PAGE and analytical gel filtration chromatography of recombinant H. pylori CCUG 17874 L-asparaginase. (A) SDS-PAGE of the recombinant enzyme purified by affinity chromatography on a Ni^⁺- Sepharose column. 1.5 μg of recombinant H. pylori L-asparaginase (lane 2) were run in parallel with prestained precision plus molecular mass markers (Biorad, lane 1) on a 12% SDS gel and stained with Coomassie Blue R-250. (B) Elution profile of L-asparaginase from a Superdex 200 10/300 GL column. The position of the peak corresponds to that of the 140 kDa E. coli L-asparaginase used as molecular mass standard. Fig. 4 Represents the steady state kinetics of recombinant H. pylori CCUG 17874 L-asparaginase as a function of L-asparagine or L-glutamine concentrations. (A) Steady state kinetics of L-asparaginase using L- asparagine (•) or L-glutamine (■ ). All experiments were performed at 37°C according to the method of Balcao (Balcao et a/., 2001 ). The kinetic vs. L- asparagine is better highlighted in the inset. (B) Hill plot of data represented in panel (A).

Fig. 5 Shows the effect of pH on the activity of L-asparaginase. 0.1 M buffers were used like the following: glycine-HCI (pH range 1-2); citric acid (3-4.5); sodium acetate (3-5.5); MES (5.5-6.5); PIPES (6-7); HEPES (7-8); tricine (8-8.5); sodium phosphate and sodium bicarbonate (9.5-10.5). The activity was assayed at 37°C using L-asparagine (•) or L-glutamine (■) as a substrate. The points are the average of at least three independent determinations. Standard Deviation is not shown for a better figure clarity. Fig. 6 Shows the thermal inactivation of recombinant H. pylori CCUG 17874 L-asparaginase. (A) Time course of inactivation of the enzyme at different temperatures: 45°C (•), 50⁰C (■), 53°C (A), 54°C (Δ), 55⁰C (T), 56°C (V), 57°C (♦). (B) Plot of the residual activities measured after heat treatment for 10 min vs. various temperatures. Fig. 7 (a) It is a cartoon representation of the active site and flexible lid loop of the calculated model of H. pylori L-asparaginase and of five L- asparaginase structures: E. coli (PDB code: 1 NNS), Pseudomonas 7A (PDB code: 1DJP), E. chrysanthemi (PDB code: 1O7J), A. glutaminasificans (PDB code: 1AGX), W. succinogenes (PDB code: 1WSA). Residue numbering according to H. pylori sequence (see Fig. 1 and Fig. 2). (b) Schematic representation of the catalytic triads involved in the reaction mechanism of asparaginase from the same model and structures of Fig. 7a. The numbering as in Fig. 7a was used. The Figure was prepared by Chimera (Pettersen et a/., 2004). Fig. 8 Represents a Western blot performed with an anti-asparaginase antibody on the subcellular fractions of H. pylori CCUG 17874. From left to right: lane 1 : pre-lytic fraction; lane 2: periplasmic fraction; lane 3 and 4: soluble and insoluble spheroplast fractions, respectively. The same protein amounts (100 μg) were loaded in each lane. Molecular mass markers (kDa) are indicated. Fig. 9 Shows the cell viability based on MTT reduction of several cell lines exposed to variable concentrations of recombinant H. pylori CCUG 17874 (a) and E. coli L-asparaginase (£>), expressed relative to the cell viability of the respective control (100%). The points represent the mean ± SD (n > 3). The viability determined for > 5 U ml^"1 was statistically significant (P<0.05, ANOVA) versus the respective control for all the cell lines tested. Fig. 10 shows the average and SD of the antibody titres detected in patients by the Diamedix kit and using L-asparaginase as an antigen. Fig. 11 Shows the scatter plot of the antibody titres detected by the Diamedix kit and L-asparaginase as an antigen. It shows a scatter chart of the antibody titres obtained with the Diamedix kit (positive samples: black diamonds; negative samples: grey triangles) and the titres of the same samples, split into the same two groups, obtained towards L-asparaginase (positive at kit: empty squares; negative at kit: black squares). Fig. 12 shows an enlarged version of the results obtained using L- asparaginase as an antigen: with black diamonds are illustrated the results obtained for the samples positive at the Diamedix kit and with empty squares those obtained for the negative ones. It shows the results obtained by testing the positive (black diamonds) and negative (empty sqares) samples with the Diamedix kit for their reactivity towards L-asparaginase.

Material and Methods Bacterial strain and culture conditions

The H. pylori CCUG 17874 strain obtained from the Culture Collection of the University Goteborg was stored at -80⁰C in 20% glycerol. After thawing, bacteria were grown on Columbia Agar plates containing 10% sheep blood, Skirrow antibiotic supplement (10 mg T¹ vancomycin, 1250 i.u. I^"1 polymixin B, 5 mg I^"1 trimethoprim Lactate; Oxoid) and Vitox (2 mg I^"1 vitamin B12, 200 mg I^"1 L-glutamine, 20 mg I^"1 adenine-SO₄, 6 mg I^'1 guanine HCI, 0.26 mg I^'1 p-aminobenzoic acid, 22 mg I^"1 L-cystine, 5 mg I^"1 NAD coenzyme I, 2 mg I^"1 cocarboxylase, 4 mg I^"1 ferric nitrate, 0.06 mg I^"1 thiamine HCI, 518 mg I^"1 cysteine HCI, 2 mg I^"1 dextrose; Oxoid) under microaerophilic conditions at 37°C for 3 d. The microaerobic atmosphere was composed by 20% CO₂, 20% H₂, 60% N₂ and 100% moisture. After a passage onto a fresh plate, bacteria were subcultured in Brucella medium (Difco) with 5% Foetal Bovine Serum (FBS, Life Tecnologies), Skirrow and Vitox, at 120 rpm, 37°C, in a thermostatic shaker under microaerophilic conditions for 24-36 h. When the culture optical density at 600 nm reached a value of 0.5 AU, bacteria from 5 ml culture were harvested by centrifugation (8000 g, 4°C, 10 min) and used for DNA extraction.

ansB gene cloning and construction of recombinant plasmids H. pylori DNA was isolated from bacterial cells by using the Dneasy tissue kit (QIAgen). The DNA encoding the putative L-asparaginase was amplified by polymerase chain reaction (PCR) using primers designed according to the sequence of the ansB gene of the H. pylori HP26695 (GenBank accession code: NC_000915), corresponding to nucleotides 777473-776481 of the minus strand of the chromosomal DNA (locus tag HP0723). The forward primer (primer A) was 5'- GGCTCATGCCATGGCTCAAAATTTACCCACCATTGC and included an Nco I site (underlined). The reverse primer (primer B) was 5¹- CATCCGCTCGAGTCATCAATACTCTTCAAACATTTCTTGG and included a Xho I site (underlined). Cycling conditions included a denaturation step at 94°C for 2 min, and 30 cycles of 94°C for 30 sec, 55°C for 30 sec, and 68⁰C for 1 min. The PCR product was A-tailed with Taq polymerase and cloned into the pCR2.1-TOPO cloning vector (Invitrogen). The insert was checked by digestion with Xho I and Nco I enzymes (New England Biolabs) and by sequencing (MWG). The recombinant plasmid containing the ansB gene of H. pylori CCUG was designated pDC1.

To express the protein, the ansB gene carried by pDC1 was amplified by PCR using cycling conditions similar to the previous ones, except for the annealing temperature (65°C), and subcloned into the pET101 vector using the Directional TOPO Expression Kit (Invitrogen). The oligonucleotides used for PCR were the following: 5'- CACCATGCATCATCACCATCACCATGACGATGACGATAAGGCTCAAA ATTTACCCACCATTGCTTT as forward primer (primer C) and primer B (see above), as reverse primer. Primer C included a 6xHis tag nucleotide sequence, an enterokinase recognition sequence, and an overlapping CACC sequence suited for TOPO cloning in the pET101 vector. The insert was checked by sequencing and the expression vector containing the desired ansB gene was named pDC2.

Sequence analysis Nucleotide sequences were assembled with Bioedit (Tippmann, 2004) and both the sequencing results and the corresponding amino acid translations were aligned with the sequences of interest using ClustalW (Myers and Miller, 1988). Identity and similarity were calculated by BLAST using the BLOUSM62 similarity matrix. Searches against the PDB database were performed by BLAST (Altschul et a/., 1990). The relevant alignment figures were generated by Multalign (Corpet, 1988).

Expression and purification of recombinant L-asparaginase

E. coli BL21 (DE3) cells transformed with pDC2 were grown at 37 ⁰C in 1 I 2xTY medium (AppliChem) containing carbenicillin (100 μg ml^"1). When the culture optical density at 600 nm reached a value of 0.6 to 0.8 AU, the expression was induced ay 20⁰C by addition of β-D-thiogalactopyranoside (IPTG) to a final concentration of 1.5 mM. After an induction of 7 h, bacteria were harvested by centrifugation and stored at -80⁰C until use. After thawing, cells were resuspended in 50 ml of 60 mM Tris-HCI pH 7.5, 100 mM NaCI (buffer A), 0.1 mM phenylmethanesulfonyl fluoride (PMFS, Sigma-Aldrich), sonicated 3 times for 1 min at 75 Watts with an Omniruptor sonicator (OMNI international) and centrifuged. The supernatant was filter-sterilised (Millex GP₁ Millipore) and loaded onto a 1 ml Ni²⁺-Sepharose column (GE Healthcare) pre-equilibrated with buffer A. The column was washed with 60 mM imidazole in buffer A, and elution was performed with 20 ml of a 60 to 150 mM imidazole gradient in buffer A. Protein fractions were analysed by 12% sodium-dodecyl-sulphate polyacrilamide gel electrophoresis (SDS-PAGE). Fractions containing the desired protein were pooled, concentrated, and dialysed against buffer A. The enzyme was stored at 4°C where it was stable for weeks.

Molecular mass determination

To determine the molecular mass of the native enzyme, the purified protein was subjected to gel filtration chromatography on a Superdex 200 10/300 GL column (GE Healthcare) equilibrated in buffer A. A total of 200 μl of purified enzyme (1 mg ml^"1) was applied to the column on a Pharmacia- LKB FPLC system (GE Healthcare) and eluted with the equilibration buffer at 0.5 ml min^"1. For column calibration the following proteins were used: lentil lectin (49 kDa), bovine serum albumin (66 kDa), E. coli L- asparaginase (140 kDa), alcohol dehydrogenase (150 kDa), β-amylase (200 kDa), apoferritin (443 kDa) and thyroglobulin (669 kDa).

Protein concentration

Protein concentration was determined by the Micro BCA™ Protein Assay Kit (Pierce), using bovine serum albumin (BSA) as a standard.

Enzyme Assay L-asparaginase activity was measured by a colorimetric method according to Wriston and Yellin (Wriston and Yellin, 1973), using a Diode array 8452A spectrophotometer (Hewlett Packard). The method was based on a stopped assay which used Nessler's reagent to determine the amount of ammonia produced during the enzymatic hydrolysis. Typically, the reaction mixture contained 100 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) pH 7.5 and 50 mM L-asparagine in a final volume of 200 μl. The reaction was started by adding the enzyme solution (1 μg), carried out at 37 ⁰C for 10 min and stopped by addition of 50 μl of 20% trichloroacetic acid solution (TCA). The ammonia produced was coupled with Nessler's reagent, and was quantitatively determined using a standard curve prepared with known concentrations of ammonium sulphate. The activity was expressed in Units (U), with one unit of L-asparaginase (U) defined as the amount of enzyme catalysing the production of 1 μmol of ammonia per min at 37 ⁰C.

Kinetic analyses

For kinetic analyses, the enzyme activity was determined in a Jasco V-550 UVΛ/IS spectrophotometer (Jasco-Europe, Cremella, Italy) at 37⁰C by means of a coupled assay with glutamate dehydrogenase, according to Balcao (Balcao et al., 2001 ), with minor modifications. The standard reaction mixture contained 50 mM HEPES, pH 7.5, 1 mM α-ketoglutarate, 20 U glutamate dehydrogenase, 0.2 mM NADH, and variable concentrations of L-asparagine or L-glutamine, in a final volume of 1 ml. The reaction was started by adding the enzyme solution (5 μg) and the activity expressed as Units, with one unit being the amount of enzyme catalysing the oxidation of 1 mmol NADH/min under the above conditions. The enzyme activity was assayed at least at 10 different concentrations of substrates. All measurements were performed in triplicate. The plot of Lineweaver-Burk was used to determine V_max and K_m values. The Hill plot was used to determine the apparent S_0.5 and ΠH values. The kinetic parameters were determined with the Enzyme Kinetic Module 1.1 (Sigma Plot, SPSS Inc.). kca_t or turnover number is the number of catalytic events per sec per active site. K_m is the substrate concentration at which the reaction velocity is half maximal. S0.5 is used for reactions displaying sigmoidal kinetics and is defined as K_m. k_ca/So_.5 is a measure of how efficiently an enzyme converts substrate to product at low substrate concentrations. Hill coefficient (ΠH) is an empirical parameter related to the degree of cooperativity; values larger than unity indicate positive cooperativity among ligand binding sites.

Thermal stability assays Thermal stability (the ability of an enzyme to withstand incubation at a given temperature) was measured by incubating the enzyme (100 μg/mL) at given temperatures in 50 mM Hepes pH 7.5. Serum bovine albumin was added to maintain the protein final concentration at 1 mg ml^"1. Samples were removed at intervals, briefly chilled on ice and assayed as described above. The relative activity was calculated as the percentage of activity of the enzyme before the incubation. t_1/2 is the time required by the enzyme to lose 50% of its activity at a given temperature; T₅₀ is the incubation temperature at which the enzyme loses 50% of its activity in 10 min.

Comparative protein structure modelling

The whole-length amino acid sequence of H. pylori CCUG L-asparaginase was submitted to 5 different homology modelling servers, each conceived to work with a different algorithm: Geno3D (Combet et al., 2002), CPH (Lund, 2002), Esypred (Lambert et al., 2002), 3Djigsaw (Contreras- Moreira and Bates, 2002) and Swissprot (Boeckmann et al., 2005). All of them are available through the Expasy server (Appel et al., 1994). The resulting structural models were visualised in Swissprot (Boeckmann et al., 2005) and superposed. Among the structural homology servers tested, the models produced by Esypred and Geno3D, based on the structures of the L-asparaginases from W. succinogenes and E. coli, turned out to be the most suitable for representing H. pylori enzyme because of their high similarity at the N-terminus. Thus, the former model and the crystallographic structure of the tetrameric E. coli L-asparaginase in complex with aspartate (PDB code: 1 NNS) were used to rebuild the homotetramer in Swissprot. The energy of the initial tetrameric model was minimized by the conjugate gradients method implemented in CNS (Brunger et al., 1998; Pai et al., 2006) and then subjected to structure idealisation using REFMAC (Winn et al., 2001) to obtain a model with ideal stereochemical parameters (i.e. absence of outliers in the Ramachandran plot), as assessed by PROCHECK (Laskowski et al., 1993). Model quality was then evaluated by PROSA 2003 (Sippl, 1993). Visual inspection of the model was carried out using the program O (Jones et al., 1991 ) and figures were prepared using the software Chimera (Pettersen et al., 2004).

Anti-L-asparaginase antibody production

For antibody production, a 6-month-old mouse (C57/B) was immunised with 30 μg recombinant H. pylori CCUG 17874 L-asparaginase + Complete Freunds Adjuvant (Pierce), followed by two boosts with 20 μg enzyme + Incomplete Freunds Adjuvant (Pierce), each 4 weeks from the previous one. Antibody titre was analysed by direct ELISA using recombinant L-asparaginase as antigen. For fusion, the mouse was euthanised and the spleen harvested. B cells were recovered from the spleen, counted and fused with an equal number of NSO mouse myeloma cells. The following day, cells were seeded in petri dishes in selective medium containing methyl-cellulose (2% w/v, Sigma) and left to grow. The colonies obtained were seeded in 96-well plates, and, when the medium turned yellow, the hybridoma supematants were tested for antibody production by ELISA. A good reactivity was obtained with an IgM antibody. The positive clone was frozen and expanded in 1 I Hybridoma-SFM medium (Invitrogen). The antibody was isolated from the culture supernatant using Protein A Sepharose (GE Healthcare). Subsequently, the anti-asparaginase antibody was subjected to biotinylation with the FluoReporter Mini-Biotin-XX Protein Labeling Kit (Molecular Probes, Invitrogen). The biotinylated antibody was then used in western blotting analysis to detect L-asparaginase in the H. pylori CCUG 17874 subcellular fractions.

Western blotting

To carry out the western blotting, proteins from different fractions and molecular weight markers (Precision Protein Standards, Biorad) were separated by a 12% SDS-PAGE in reducing conditions and transferred onto a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with 5% (w/v) milk powder in PBS containing 0.05% Tween (PBS- T) and incubated with the mouse monoclonal biotinylated anti- Asparaginase antibody in PBS-T, 1 % BSA for 1 h. After 3 x 5 min washings in PBS-T, blots were incubated with a streptavidin-horseradish peroxidase conjugate (GE Healthcare) in PBS-T for 45 min, washed 3 times, and visualised by ECL (GE Healthcare).

Subcellular fractions preparation

For subcellular fractionation, the H. pylori CCUG 17874 cell pellet obtained by centrifugation from a 50 ml bacterial culture was washed in 10 ml PBS, resuspended in 1.5 ml of 10 mM Tris HCI pH 7.6, 20% sucrose and after a 5 min incubation on ice mixed with 50 μl of 0.5 M EDTA₁ pH 8.0. After a 10 min incubation at high osmotic strength and subsequent centrifugation, the supernatant containing proteins leaked out of the cells or located at or near the cell surface was collected (fraction 1 or pre-lytic), whereas the cells were converted into spheroplasts by osmotic shock, shifting them to a medium of low osmotic strength. Briefly, the cell pellet was resuspended in 1 ml cold water, vortexed, incubated on ice for 10 min, and centrifuged. The supernatant containing the periplasmic fraction was collected (fraction 2), whereas the pellet containing the spheroplasts was resuspended in 500 μl 1 M Tris HCI, pH 7.6, vortexed, incubated on ice for 30 min and centrifuged. The supernatant corresponding to the spheroplast soluble fraction was recovered (fraction 3), and the pellet containing the spheroplast insoluble fraction was resuspended in 500 μl 50 mM Tris HCI, pH 8.0, 2 mM EDTA, 0.1 mM DTT, 5% (v/v) glycerol, (fraction 4).

Cell culture and MTT assays

Cells from different cell lines were routinely grown at 37⁰C in DMEM/Ham's nutrient mixture F-12 (MKN7, MKN28 and MKN74); RPMI 1640 (AGS, HL60 and MOLT-4) and E-MEM (HDF) containing 100 IU ml^"1 penicillin and 100 μg ml^"1 streptomycin, and supplemented with 10% foetal calf serum, in a 5% CO2 humidified atmosphere. For the viability test, cells were inoculated in 96-well tissue culture plates at 4000/well in 100 μl medium and incubated at 37⁰C with increasing concentrations of recombinant H. pylori L-asparaginase or E. coli type Il L-asparaginase (Sigma) for 24 h. At the end of treatment, cell survival was determined by MTT assay (Mosmann, 1983). In order to do this, 10 μl of a 5 mg ml^"1 solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) in PBS were added to each well, and, after 2 h incubation at 37⁰C and subsequent centrifugation, 100 μl DMSO were added to lyse the cells and solubilise the formazan crystals (reduced MTT) produced by the mitocondrial dehydrogenase activity. The absorbance of the formazan solution was then measured at 570 nm. Viability values were compared by ANOVA and Tukey test. The results were also expressed as IC₅₀, that is the L-asparaginase concentration inducing 50% cytotoxicity. Example 1

Characterization of L-asparaginase from Helicobacter pylori CCUG

17874

1a) Molecular cloning of H. pylori CCUG 17874 ansB gene and sequence alignments

With the aim to investigate the putative H. pylori CCUG 17874 L- asparaginase, the relevant DNA fragment was amplified by PCR using primers designed according to the sequence of the ansB gene of the H. pylori strain HP26695 (Genbank accession code: NC_000915), annotated as probable L-asparaginase by sequence similarity (locus tag: HP0723). The PCR product was inserted into the pCR2.1-TOPO vector, thus generating the pDC1 construct, and sequenced. The cloned gene consisted of 993 bp, corresponding to a protein of 330 amino acid residues. The nucleotide resulted identical in 4 clones obtained from independent PCR amplifications (Fig. 1 ). This sequence was compared with those encompassing the ansB gene of three different H. pylori strains, i.e., HP26695, J99 and HPAG1 (Genbank accession codes: NC_000915, NC_000921 , and NC_008086, respectively). An identity of 92.00% was consistently found. The deduced amino acid sequence of this DNA fragment was then aligned with those of the above mentioned H. pylori strains (Genbank accession codes: NP_207517, NP_223379 and YP_627449, respectively), and an identity of 93.00%, 91.00% and 93.00%, respectively, was calculated (Fig. 1 ). Finally, the full-length amino acid sequence was aligned with those of bacterial L-asparaginases whose structures have been deposited in the PDB database (Fig. 2), i.e., Wolinella succinogenes {W. succinogenes), E. coli, Pseudomonas 7A, E. chrysanthemi and Acinetobacter glutaminasificans (A. glutaminasificans), revealing a similarity of 29.09%, 18.78%, 26.36%, 46.67%, 22.96%, respectively, as calculated by BLAST, BLOUSM62. It is worth noting that the low similarity displayed by the putative H. pylori L-asparaginase with respect to the other corresponding bacterial enzymes was not a novelty to this family of enzymes (Kotzia and Labrou, 2007). On the other hand, the amino acid residues belonging to the two catalytic triads and absolutely conserved in all the known bacterial L-asparaginases (Sanches et al., 2007) were also present in the H. pylori CCUG 17874 enzyme, once more confirming their crucial role (Fig. 1 and Fig. 2).

1b) Protein expression and purification

In order to obtain the L-asparaginase in large quantity and facilitate its purification, the H. pylori CCUG 17874 ansB gene endowed with the nucleotide sequence for an N-terminal 6xHis tag was subcloned into the pET101/D-TOPO vector (see Materials and Methods). The resulting recombinant expression vector, designated pDC2, was used to transform E. coli BL21(DE3) cells. The highest level of soluble protein was gained on induction with IPTG at a final concentration of 1.5 mM at 20⁰C for 7 h. The expressed protein was purified to homogeneity by affinity chromatography on a Ni²⁺-Sepharose column. The elution of the protein from the column was performed using a 60 to 150 mM imidazole gradient. The purity of the enzyme preparation was evaluated on a 12% SDS- PAGE. A single band was observed on the gel, corresponding to a molecular mass of approximately 37 kDa (Fig. 3a). This value was in good agreement with that predicted from the amino acid sequence for the recombinant protein (36,915.93 Da). Typically, the protein yield resulted to be about 1 ± 0.2 mg per litre of culture. The enzyme activity was linear with the protein concentration, and the specific activity was 31.2 U mg^"1. The enzyme was also active towards L-glutamine, although with a much lower catalytic efficiency (see below, Kinetic Properties, 1d)). Removal of the 6xHis tag by incubation with enterokinase did not significantly affect activity (data not shown).

1c) Assessment of the oligomeric state of the enzyme Nearly all members of the L-asparaginase protein family are homotetramers. In order to ascertain the oligomeric state of the H. pylori CCUG 17874 L-asparaginase, the purified protein was subjected to analytical gel filtration chromatography on a Superdex 200 column. The enzyme eluted from the column as a single peak, symmetric in shape, at a position corresponding to that of the E. coli L-asparaginase used as a 140 kDa standard (Fig. 3b). Thus, on the basis of the value of the molecular mass displayed on SDS-PAGE, the recombinant protein proved to be a tetramer of identical subunits. In this respect, the enzyme displayed molecular characteristics similar to most known bacterial asparaginases (Sanches ef a/., 2007).

1d) Kinetic properties of the recombinant L-asparaginase from H. pylori CCUG 17874.

Steady-state kinetics of the enzyme as a function of substrate concentration To ascertain to which group of L-asparaginases the H. pylori CCUG 17874 enzyme could be assigned, a kinetic analysis was carried out using either L-asparagine or L-glutamine as a substrate. Steady state kinetics were determined as reported under Experimental Procedures. The main kinetic parameters obtained are reported in Table 1. The H. pylori CCUG 17874 enzyme exhibited a hyperbolic response with respect to L-asparagine, and, surprisingly, a sigmoidal behaviour towards L- glutamine (Fig. 4a). The k∞_t values were comparable (19.26 s^'\ and 22.10 s^"1, respectively), but the apparent affinity towards L-asparagine was 2 orders of magnitude higher than that versus L-glutamine. In fact, as determined from Hill plot (Fig. 4b), the concentration of substrate required for half maximal velocity (S₀.₅) was 0.29 mM in the case of L-asparagine and 46.4 mM in the case of L-glutamine. As a consequence, the catalytic efficiency exhibited by the enzyme vs. L-asparagine was approximately 140 folds higher than that vs. L-glutamine. These findings allowed us to assign this novel enzyme to the group of type Il L-asparaginases. However, the fact that the enzyme showed a typical homotropic response to L-glutamine with a Hill coefficient (n_H) of 2 was puzzling. Thus, in order to shed light on a potential allosteric effector, we investigated a number of different compounds. Unfortunately, none of the molecules or ions assayed modified substantially the sigmoidal curve vs. L-glutamine. Only a moderate activation was observed in the presence of 100 mM potassium phosphate (data not shown).

Table 1 Kinetic parameters of recombinant H. pylori CCUG 17874 L- asparaginase

0.29 ± 1.00 ±

L-asparagine 19.26 ± 0.56 66.41 0.03 0.06

46.40 ± 2.00 ± L-glutamine 22.10 ± 1.39 0.48 4.02 0.10

Results are means ± SE for 3 determinations from 3 different preparations

1 e) Activity dependence on DH

The enzyme activity as a function of pH was measured at saturating concentrations of L-asparagine or L-glutamine, using the method based on Nessler's reagent (Wriston and YeIHn, 1973) and/or the method of Balcao (Balcao et a/., 2001 ; Fig. 5). The latter method could not be applied to assays performed at pH lower than 7, the glutamate dehydrogenase used as coupled enzyme being inactive at acidic pH values. When the enzyme was assayed for its glutaminase activity, the pH-rate profile approximated a bell-shaped curve, with a maximum activity at a pH value of 7.5 and a lack of activity below pH 5. When L-asparagine was used as a substrate, a sigmoidal pH-rate profile was observed, with a broad pH optimum in the pH range 7.0-10.0. In this case, even below pH 4 the enzyme displayed activity, although to a lower extent (less than 20%). As a whole, these data suggest that the enzyme adopts a different strategy to convert the two substrates into their corresponding acids and ammonia.

1f) Thermal stability properties To evaluate the stability of the H. pylori L-asparaginase, the purified enzyme was subjected to heat inactivation assays. It resulted to be quite stable, retaining full activity after 2 h incubation at 45°C. At 50⁰C it still preserved 50% of its activity after a treatment of 36 min, whereas it turned off completely in a few min when the temperature was raised above 55°C (ti_/2 at 55°C: 3 min; Fig. 6a). A more comprehensive picture of the behaviour of H. pylori L-asparaginase with respect to heat was obtained subjecting the enzyme to a wider range of temperatures within 10 min. The enzyme turned out to be absolutely stable up to 50⁰C. The T₅₀ resulted 53°C (Fig. 6b). These data suggest that the H. pylori CCUG 17874 enzyme may be suitable as a therapeutic agent.

1q) Structural modelling

In order to produce a preliminary evaluation of the configuration of the enzyme active site, a model of H. pylori CCUG 17874 L-asparaginase was built by homology modelling (see Materials and Method). PROSA 2003 Z- score, calculated for the energy minimised and idealised homotetrameric model, was -9.15, indicating a model of good quality (Sippl, 1993). Fig. 7 depicts the model of the active site of H. pylori L-asparaginase overlapped on 5 known L-asparaginase structures (Sanches et a/., 2007). All the residues of the two catalytic triads of the generated model perfectly overlap onto those of the known structures, except for Glu289. In this respect, the neighbouring H. pylori L-asparaginase sequence, like in E. chrysantemi, is peculiar with respect to the other bacterial species analysed for its shorter length (Fig. 2). This suggests that the role for the alternative position assumed by Glu289 might be relevant in the determination of the activity of the enzyme and would be an interesting issue to be clarified by further structural studies. 1h) Sub-cellular localisation of L-asparaqinase

The subcellular fractions of H. pylori CCUG 17874 prepared as reported under Experimental procedures were subjected to western blotting analysis to localise L-asparaginase. A monoclonal antibody specifically raised towards H. pylori CCUG 17874 L-asparaginase (Materials and Methods) allowed us to evidence two L-asparaginase forms with different molecular masses in all the three subcellular fractions analysed, that is the periplasmic and the spheroplast soluble and insoluble fractions (Fig. 8, lanes 2, 3 and 4). In all the fractions analysed, the higher molecular mass form (approximately 39 kDa) was less represented (40%) than the lower one (about 37 kDa, 60%). The difference between the two molecular masses (2 kDa) agrees with the approximate one calculated for the cleavable signal sequence predicted both by the program SignalP 3.0 (Bendtsen et al., 2004) and PSORTb (Gardy et a/., 2005). Interestingly, the pre-lytic fraction seemed to include, although less represented, only the 37 kDa-form (Fig. 8, lane 1), indicating that, in appropriate conditions, this form of the enzyme is mobile and free to leak through the outer bacterial membrane, while the 39 kDa-form might be membrane associated, as predicted by the BII server (http://protein.bii.a- star.edu.sg/sgi-bin/localization/gram-negative).

EXAMPLE 2 Cytotoxicity of L-asparaginase

To investigate L-asparaginase cytotoxicity in vitro, several cell lines were tested. Since a normal human gastric cell lines is not available, the following human gastric epithelial cell lines were used: AGS, derived from gastric adenocarcinoma; MKN28, derived from a human gastric tubular adenocarcinoma and showing moderate gastric-type differentiation; MKN7, derived from a well-differentiated tubular adenocarcinoma; MKN74, originating from a moderately differentiated tubular adenocarcinoma. Leukemic cells (HL60 and MOLT-4) and normal human diploid embryonic fibroblasts (HDF) were also tested. The effect of variable concentrations of recombinant H. pylori CCUG 17874 L-asparaginase on cell survival, as judged by a standard MTT assay (see Material and Methods), is shown in Fig. 9a whereas the relative IC₅₀ values are reported in Table 2. The viability of the cell lines investigated were heavily affected by the presence of the enzyme in their culture medium, except for normal HDF (IC₅₀ up to 4 orders of magnitude higher). Among the affected cell lines, AGS and MKN28 gastric epithelial cells and HL60 leukemic cells turned out to be the most impaired, whereas the MOLT-4 cell line resulted more resistant (IC50 forty times higher than AGS cells). These data underline the importance of the genotype of the cell line tested, and suggest a potentially variable effect on different tissues and cell lineages in vivo, both normal and tumoral. The effect of E. coli L-asparaginase is reported for comparison (Fig. 9b and Table 2).

Table 2 IC₅₀ (U ml^"1) of H. pylori and E. coli L-asparaginase towards different cell lines.

(N.S.: Not Sensitive, IC₅₀ >15000 U ml^"1)

EXAMPLE 3 Immunologic diagnostic assay using the recombinant protein produced according to the present invention

3a) Selection of patients

The serum of each patient was analysed for diagnostic purposes using the commercial kit by Diamedix. The remaining serum was then transferred to the Laboratory of General Pathology of the Department of Experimental Medicine of the University of Pavia, where both repetition of the commercial test, to check serum preservation during shipping, and an ELISA test using recombinant L-asparaginase from H. pylori as an antigen were performed. The experimental procedures are detailed below.

3b) ELISA assay for the determination of the serum antibody titre using a commercial kit

To determine the antibody titre, the H. pylori IgG Enzyme Immunoassay Test kit by Diamedix, an immunoenzymatic assay with recombinant antigens to detect IgG against H. pylori in human serum and plasma, was used. The microplate included in the kit is coated with bacterial antigens produced in recombinant form, among which CagA and VacA. For the test, the samples and standard were prepared for calibration diluting them 1 :100 in 1 ml sample diluent. The pre-coated microplate was removed from the pressure-sealed bag and 100 μl of samples and standards were pipetted in each well of the microplate. Once taped over with sealing tape, the microplate was incubated at 37°C for 1 h. Then, the wells were emptied completely and washed 4 times with 300 μl washing solution, removing all the solution between one washing step and the next and tapping over a paper towel after the last wash. 100 μl of peroxidase conjugated antibody were pipetted in each well and, after sealing, the microplate was incubated at 37°C for 30 min. After washing like described above, 100 μl substrate were added to each well and left to incubate far from direct sunlight at room temperature for 30 min. The reaction was stopped with 100 μl stop solution. The reading was performed at 450 nm in a microplate reader (Biorad) within 60 min of stopping the reaction and using air as blank.

Quality control samples have been used, in particular: a negative control, with an absorbance lower than or equal to 0.150; a difference between the cutoff control and the negative control higher than or equal to 0.050 and a similar difference between the positive control and the cutoff control higher than or equal to 0.300. For the quantification of results, the following formula is used:

U/ml = (sample absorbance/absorbance cutoff) x 20 The results are interpreted like the following: The test is positive if >24 U/ml The test is negative if <20 U/ml The test is borderline if comprised between 20 and 24 U/ml

The test has the following performance characteristics: Specificity: 98%

Sensitivity: gastritis: >97% carcinoma: 100% 3c) ELISA assay for the determination of the serum antibody titre using the recombinant L-asparaqinase from H. pylori CCUG 17874 of the invention A 96-well plate (Pierce) was coated with recombinant H. pylori L- asparaginase by incubating each well with 100 μl protein, produced like described in the previous sections and resuspended at the concentration of 50 μg/ml in PBS. The incubation was performed in a humid chamber at 4°C for 16 h, after which the solution was removed and the wells washed 4 times with 300 μl washing buffer. The patients' sera, diluted as described above for the commercial assay, were placed in the wells of the coated microplate. The following steps were performed in parallel with the commercial test and exactly like described above, including the quality control samples. Calculations and interpretation of the results were also performed as described above.

Detection of IgM antibodies in patients' serum can easily be performed replacing a peroxidase conjugated anti-human IgG antibody with a peroxidase conjugated anti-human IgM antibody.

3d) Statistical analysis

The data obtained from the ELISA assays were analysed by McNemar Chi Square Test to compare paired population proportions of positive results. The Diamedix commercial kit was used as gold standard. The results of ELISA assays are showed in Table 3, which reports the data obtained from the 24 patients analysed. Out of these, 13 were positive (+) and 11 negative (-) according to the Diamedix. In the same Table 3, the antibody titres obtained towards recombinant L-asparaginase are reported. It is possible to observe several differences between the two data series, with some positive patients resulting negative or borderline at the test performed with L-asparaginase, and, vice versa, negative patients showing positive or borderline reactivity towards L-asparaginase. Table 3. Antibody titres obtained with Diamedix kit and using recombinant L-asparaginase as an antigen.

Legend: (+): positive; (-): negative; between parenthesis: borderline.

Table 4 below summarises the data concerning the antibody titres for different patients¹ groups (positive, negative and borderline), while Fig. 10 illustrates the relevant averages and Standard Deviation. The antibody titre detected by the Diamedix kit is on average higher than that detected by the assay using recombinant L-asparaginase.

Table 4. Statistics of the antibody titres obtained with the Diamedix kit and using recombinant L-asparaginase as an antigen.

Table 5 below reports the data used for the calculation of McNemar Chi Square Test to compare the results based on L-asparaginase with those based on the Diamedix kit. The McNemar Chi Square Test value is 0.227, showing that proportions of positive results for the two ELISA tests were not significantly different and indicating an essential equivalence between them. The sensitivity of the test was 0.63 and its specificity 0.50, with an efficacy of 54,17%. The predictive value was 0.38. Most importantly, the preliminary data here illustrated also suggest that L-asparaginase might allow the identification of a 25% portion of the patients negative at the Diamedix kit, indicating that the new antigen represents a valuable contribution to the improvement of the kit sensitivity.

Table 5 Cross classification of the Diamedix kit and the L-asparaginase based test. McNemar Chi Square Test (P=O.227).

EXAMPLE 4

Application of the L-asparaginase of the invention to starch-based foods

A suitable amount of the L-asparaginase from Helicobacter pylori CCUG 17874 of the invention is dispersed in a solution. The starch-based food is immersed in said solution for a suitable period of time; after that it is removed from the solution, dried and processed at a temperature above 120⁰C.

The amount of acrylamide produced can be measured by GS- chromatography and is expected to be lower than the amount of acrylamide produced in cases wherein L-asparaginase is not .

References

Aghaiypour, K., Wlodawer, A., and Lubkowski, J. (2001) Structural basis for the activity and substrate specificity of Erwinia chrysanthemi L-asparaginase. Biochemistry 40: 5655-5664. Albertsen, B.K., Jakobsen, P., Schroder, H., Schmiegelow, K., and Carlsen, N.T. (2001) Pharmacokinetics of Erwinia asparaginase after intravenous and intramuscular administration. Cancer Chemother Pharmacol 48: 77-82. Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, DJ. (1990) Basic local alignment search tool. J MoI Biol 215: 403-410. Appel, R.D., Bairoch, A., and Hochstrasser, D.F. (1994) A new generation of information retrieval tools for biologists: the example of the ExPASy WWW server. Trends Biochem Sci 19: 258-260. Avramis, V.I., and Tiwari, P.N. (2006) Asparaginase (native ASNase or pegylated

ASN ase) in the treatment of acute lymphoblastic leukemia. Int J Nanomedicine 1 : 241 -254.

Bahari, H.M., Ross, I.N., and Turnberg, L.A. (1982) Demonstration of a pH gradient across the mucus layer on the surface of human gastric mucosa in vitro. Gut 23: 513-516.

Balcao, V.M., Mateo, C, Fernandez-Lafuente, R., Malcata, F.X., and Guisan, J.M. (2001) Structural and functional stabilization of L-asparaginase via multisubunit immobilization onto highly activated supports. Biotechnol Prog 17: 537-542. Bauerfeind, P., and Wirth, H.P. (1997) [Peptic ulcer, Helicobacter pylori]. Ther

Umsch 54: 624-628. Bendtsen, J.D., Nielsen, H., von Heijne, G., and Brunak, S. (2004) Improved prediction of signal peptides: SignalP 3.0. JMoI Biol 340: 783-795. Boeckmann, B., Blatter, M.C., Famiglietti, L., Hinz, U., Lane, L., Roechert, B., and Bairoch, A. (2005) Protein variety and functional diversity: Swiss-Prot annotation in its biological context. C R Biol 328: 882-899. Boyse, E. A., Old, LJ., Campbell, H. A., and Mashburn, L.T. (1967) Suppression of murine leukemias by L-asparaginase. Incidence of sensitivity among leukemias of various types: comparative inhibitory activities of guinea pig serum L-asparaginase and Escherichia coli L-asparaginase. J Exp Med 125: 17-31. Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse- Kunstleve, R. W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R. J., Rice, L.M., Simonson, T., and Warren, G.L. (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54: 905-921.

Bush, L.A., Herr, J.C., Wolkowicz, M., Sherman, N.E., Shore, A., and Flickinger, CJ. (2002) A novel asparaginase-like protein is a sperm autoantigen in rats. MoI Reprod Dev 62: 233-247.

Cammack, K.A., Marlborough, D.I., and Miller, D.S. (1972) Physical properties and subunit structure of L-asparaginase isolated from Erwinia carotovora.

Biochem J 126: 361-379. Carter, P., and Wells, J.A. (1988) Dissecting the catalytic triad of a serine protease. Nature 332: 564-568. Chang, T.M. (1984) Artificial cells in medicine and biotechnology. Appl Biochem

Biotechnol 10: 5-24. Combet, C, Jambon, M., Deleage, G., and Geourjon, C. (2002) Geno3D: automatic comparative molecular modelling of protein. Bioinformatics 18: 213-214. Contreras-Moreira, B., and Bates, P.A. (2002) Domain fishing: a first step in protein comparative modelling. Bioinformatics 18: 1141-1142. Corpet, F. (1988) Multiple sequence alignment with hierarchical clustering.

Nucleic Acids Res 16: 10881-10890. Derst, C, Henseling, J. and Rohm K.H. (2000) Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248. Protein Sd. 9: 2009-17.

Distasio, J.A., Niederman, R. A., Kafkewitz, D. and Goodman D. (1976)

Purification and characterization of L-asparaginase with anti-lymphoma activity from Vibrio succinogenes. J Biol Chem 251: 6929-33. Distasio, J. A., Salazar, A.M., Nadji, M., Durden, D. L. (1982) Glutaminase-free asparaginase from vibrio succinogenes: an antilymphoma enzyme lacking hepatotoxicity. Int J Cancer 30: 343-347. Dodson, G., and Wlodawer, A. (1998) Catalytic triads and their relatives. Trends

Biochem Sci 23: 347-352.

Duval, M., Suciu, S., Ferster, A., Rialland, X., Nelken, B., Lutz, P., Benoit, Y., Robert, A., Manel, A.M., Vilmer, E., Otten, J., and Philippe, N. (2002)

Comparison of Escherichia coli-asparaginase with Erwinia-asparaginase in the treatment of childhood lymphoid malignancies: results of a randomized European Organisation for Research and Treatment of Cancer-Children's Leukemia Group phase 3 trial. Blood 99: 2734-2739. Eaton, K.A., Brooks, C.L., Morgan, D.R., and Krakowka, S. (1991) Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic piglets. Infect Immun 59: 2470-2475. Ehrman, M., Cedar, H., and Schwartz, J.H. (1971) L-asparaginase II of

Escherichia coli. Studies on the enzymatic mechanism of action. J Biol Chem 246: 88-94.

El-Bessoumy, A.A., Sarhan, M., and Mansour, J. (2004) Production, isolation, and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation. J Biochem MoI Biol 37: 387-393. Femandes, A.I., and Gregoriadis, G. (1997) Polysialylated asparaginase: preparation, activity and pharmacokinetics. Biochim Biophys Acta 1341:

26-34.

Fiocca, R., Necchi, V., Sommi, P., Ricci, V., Telford, J., Cover, T.L., and Solcia, E. (1999) Release of Helicobacter pylori vacuolating cytotoxin by both a specific secretion pathway and budding of outer membrane vesicles. Uptake of released toxin and vesicles by gastric epithelium. J Pathol 188:

220-226.

Gallagher M.P., Marshall R.D. and Wilson R. (1989) Asparaginase as a drug for treatment of acute lymphoblastic leukaemia. Essays Biochem. 24:1-40. Gardy, J.L., Laird, M.R., Chen, F., Rey, S., Walsh, C.J., Ester, M., and Brinkman, F. S. (2005) PSORTb v.2.0: expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis. Bioinformatics 21: 617-623. Ho, P.P., Milikin, E.B., Bobbitt, J.L., Grinnan, E.L., Burck, P.J., Frank, B.H., Boeck, L.D., and Squires, R.W. (1970) Crystalline L-asparaginase from Escherichia coli B. I. Purification and chemical characterization. J Biol Chem 245: 3708-3715.

Howard, J.B., and Carpenter, F.H. (1972) L-asparaginase from Erwinia carotovora. Substrate specificity and enzymatic properties. J Biol Chem

241: 1020-1030.

Jones, T.A., Zou, J.Y., Cowan, S.W., and Kjeldgaard, M. (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta CrystallogrA 47 ( Pt 2): 110-119. Jonsson, K., Guo, B.P., Monstein, H. J., Mekalanos, JJ. , and Kronvall, G. (2004) Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins. Proc Natl Acad Sci USA 101: 1852-1857.

Kanehisa, M., Goto, S., Hattori, M., Aoki-Kinoshita, K.F., Itoh, M., Kawashima, S., Katayama, T., Araki, M., and Hirakawa, M. (2006) From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res 34: D354-357.

Keating, M. J., Holmes, R., Lerner, S., and Ho, D. H. (1993) L-asparaginase and PEG asparaginase— past, present, and future. Leuk Lymphoma 10 Suppl: 153-157.

Kotzia, G.A., and Labrou, N.E. (2007) L-Asparaginase from Erwinia

Chrysanthemi 3937: cloning, expression and characterization. J Biotechnol 127: 657-669.

Krasotkina, J., Borisova, A.A., Gervaziev, Y.V., and Sokolov, N.N. (2004) One- step purification and kinetic properties of the recombinant L-asparaginase from Erwinia carotovora. Biotechnol Appl Biochem 39: 215-221. Lambert, C, Leonard, N., De Bolle, X., and Depiereux, E. (2002) ESyPred3D:

Prediction of proteins 3D structures. Bioinformatics 18: 1250-1256. Laskowski, R., MacArthur, M., Moss DS, and JM, T. (1993) PROCHECK: A program to check the stereochemical quality of protein structures. J. Appl.

Cryst 26: 283-291.

Lubkowski, J., Palm, G.J., Gilliland, G.L., Derst, C, Rohm, K.H., and Wlodawer, A. (1996) Crystal structure and amino acid sequence of Wolinella succinogenes L-asparaginase. Eur J Biochem 241: 201-207. Lund, O., Nielsen, M., Lundergaard P. (2002) CPHmodels 2.0:X3M a Computer Program to Extract 3D Models. Worning Abstracts at the CASAP5 confer enceA 102. Makristathis, A., Pasching, E., Schutze, K., Wimmer, M., Rotter, M.L., and

Hirschl, A.M. (1998a) Detection of Helicobacter pylori in stool specimens by PCR and antigen enzyme immunoassay. J Clin Microbiol 36: 2772-

2774.

Makristathis, A., Rokita, E., Labigne, A., Willinger, B., Rotter, M.L., and Hirschl, A.M. (1998b) Highly significant role of Helicobacter pylori urease in phagocytosis and production of oxygen metabolites by human granulocytes. In J Infect Dis. Vol. 177, pp. 803-806. Mendz, G.L., and Hazell, S. L. (1995) Aminoacid utilization by Helicobacter pylori. Int J Biochem Cell Biol 27: 1085-1093. Merrell, D.S., Goodrich, ML., Otto, G., Tompkins, L.S., and Falkow, S. (2003) pH-regulated gene expression of the gastric pathogen Helicobacter pylori. Infect Immun 71: 3529-3539. Mickalska K., and Jaskolski M. (2006) Structural aspects of L-asparaginases, their friends and relations. Acta Biochim Pol 53:627-640. Miller, M., Rao, J.K., Wlodawer, A., and Gribskov, M.R. (1993) A left-handed crossover involved in amidohydrolase catalysis. Crystal structure of Erwinia chrysanthemi L- asparaginase with bound L-aspartate. FEBS Lett 328: 275-279.

Moola, Z.B., Scawen, M.D., Atkinson, T., and Nicholls, DJ. (1994) Erwinia chrysanthemi L-asparaginase: epitope mapping and production of antigenically modified enzymes. Biochem J 302 ( Pt 3): 921-927. Mosmann, T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55-63. Myers, E.W., and Miller, W. (1988) Optimal alignments in linear space. Comput

Appl Biosci 4: 11-17. Nagata, Y., Sato, T., Enomoto, N., Ishii, Y., Sasaki, K., and Yamada, T. (2007)

High concentrations of D-amino acids in human gastric juice. Amino Acids 32: 137-140. Necchi, V., Candusso, M.E., Tava, F., Luinetti, O., Ventura, U., Fiocca, R., Ricci, V., and Solcia, E. (2007) Intracellular, intercellular, and stromal invasion of gastric mucosa, preneoplastic lesions, and cancer by Helicobacter pylori. Gastroenterology 132: 1009-1023.

Ollenschlager, G., Roth, E., Linkesch, W., Jansen, S., Simmel, A. and Mόdder B. (1988) Asparaginase-induced derangements of glutamine metabolism: the pathogenetic basis for some drug-related side-effects. Eur J Clin Invest. 18: 512-516. Pai, R., Sacchettini, J., and Ioerger, T. (2006) Identifying non-crystallographic symmetry in protein electron-density maps: a feature-based approach. Acta Crystallogr D Biol Crystallogr 62: 1012-1021.

Pettersen, E.F., Goddard, T.D., Huang, CC, Couch, G.S., Greenblatt, D.M., Meng, E.C, and Ferrin, T.E. (2004) UCSF Chimera~a visualization system for exploratory research and analysis. J Comput Chem 25: 1605- 1612. Poole, B., and Ohkuma, S. (1981) Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages. J Cell Biol 90: 665-669. Reinert, R. B., Oberle, L. M., Wek, S. A., Bunpo, P., Wang, X. P., Mileva, I.,

Goodwin, L. O., Aldrich, C J., Durden, D. L., McNurlan, M. A., Wek, R. C and Anthony, T. G. (2006) Role of glutamine depletion in directing tissue-specific nutrient stress responses to L-asparaginase. J Biol Chem

281:31222-31233.

Rohm, K.H., and Van Etten, R.L. (1986) Catalysis by hog-kidney aminoacylase does not involve a covalent intermediate. Eur J Biochem 160: 327-332. Sanches, M., Krauchenko, S. and Polikarpov, I. (2007) Structure, Substrate

Complexation and reaction Mechanism of Bacterial Asparaginases.

Current Chemical Biology 1: 75-86.

Sippl, M.J. (1993) Recognition of errors in three-dimensional structures of proteins. Proteins 17: 355-362.

Smoot, D.T., Mobley, H.L., Chippendale, G.R., Lewison, J.F., and Resau, J.H.

(1990) Helicobacter pylori urease activity is toxic to human gastric epithelial cells. Infect Immun 58: 1992-1994.

Sommi, P., Ricci, V., Fiocca, R., Romano, M., Ivey, KJ. , Cova, E., Solcia, E., and Ventura, U. ( 1996) Significance of ammonia in the genesis of gastric epithelial lesions induced by Helicobacter pylori: an in vitro study with different bacterial strains and urea concentrations. Digestion 57: 299-304. Stark, R.M., Suleiman, M.S., Hassan, I.J., Greenman, J., and Millar, M.R. (1997)

Amino acid utilisation and deamination of glutamine and asparagjne by Helicobacter pylori. J Med Microbiol 46: 793-800.

Stecher, A.L., de Deus, P.M., Polikarpov, I., and Abrahao-Neto, J. (1999)

Stability of L-asparaginase: an enzyme used in leukemia treatment. Pharm

Acta HeIv 74: 1-9.

Swain, A.L., Jaskolski, M., Housset, D., Rao, J.K., and Wlodawer, A. (1993) Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy. Proc Natl Acad Sci US A 90: 1474-1478. Tippmann, H.F. (2004) Analysis for free: comparing programs for sequence analysis. Brief Bioinform 5: 82-87.

Wacker, P., Land, V.J., Camitta, B.M., Kurtzberg, J., Pullen, J., Harris, M.B., and Shuster, J.J. (2007) Allergic Reactions to E. coli L- Asparaginase Do Not

Affect Outcome in Childhood B-precursor Acute Lymphoblastic

Leukemia: A Children's Oncology Group Study. J Pediatr Hematol Oncol

29: 627-632.

Wileman, T.E., Foster, R.L., and Elliott, P.N. (1986) Soluble asparaginase-dextran conjugates show increased circulatory persistence and lowered antigen reactivity. J Pharm Pharmacol 38: 264-271. Winn, M.D., Isupov, M.N., and Murshudov, G.N. (2001) Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta

Crystallogr D Biol Crystallogr 57: 122-133. Wriston, J.C., Jr., and Yellin, T.O. (1973) L-asparaginase: a review. Adv Enzymol

Relat Areas MoI Biol 39: 185-248. Xu, J.K., Goodwin, C.S., Cooper, M., and Robinson, J. (1990) Intracellular vacuolization caused by the urease of Helicobacter pylori. J Infect Dis

161: 1302-1304. Yao, M., Yasutake, Y., Morita, H., and Tanaka, I. (2005) Structure of the type I L- asparaginase from the hyperthermophilic archaeon Pyrococcus horikoshii at 2.16 angstroms resolution. Acta Crystallogr D Biol Crystallogr 61: 294-

301.

Yun, M.K., Nourse, A., White, S.W., Rock, CO., and Heath, RJ. (2007) Crystal structure and allosteric regulation of the cytoplasmic Escherichia coli L- asparaginase I. JMoI Biol 369: 794-811.

Claims

CLAIMS:

1. An isolated nucleic acid encoding the L-asparaginase from Helicobacter pylori CCUG 17874 and having the sequence of Seq. ID n. 1 , its complementary or hybridizing sequences, and any fragments thereof.

2. The isolated nucleic acid of claim 1 , which encodes for the L- asparaginase from Helicobacter pylori CCUG 17874 having the amino acidic sequence corresponding to Seq. ID n. 2.

3. An expression vector comprising any one of the isolated nucleic acids of claim 1 or 2.

4. A host cell comprising the expression vector of claim 3.

5. An Escherichia coli host cell comprising the expression vector of claim 3.

6. An isolated amino acidic sequence of L-asparaginase from Helicobacter pylori CCUG 17874 having the sequence of Seq. ID n.

2, and any fragments thereof.

7. A process for the preparation of recombinant L-asparaginase from Helicobacter pylori CCUG 17874, comprising the steps of: a) culturing the Escherichia coli cells of claim 5 allowing them to express the L-asparaginase of claim 6 in an appropriate medium culture; and b) recovering the L-asparaginase excreted by said Escherichia coli cells and purifying it.

8. The process according to claim 7 for the preparation of recombinant L-asparaginase from Helicobacter pylori CCUG 17874 having the amino acidic sequence of Seq. ID n. 2.

9. A process for preparing monoclonal antibodies against the L- asparaginase of claim 6, comprising the steps of: a) immunizing a mouse with an amount of the L-asparaginase of claim 6, suitable to elicit an immune response; b) isolating immunized B-cells from said animal; and c) fusing the recovered B-cells under appropriate fusion conditions with an immortalizing cell line to obtain hybridoma cells; d) screening the hybridomas and selecting the L-asparaginase producing hybridoma cells; e) expanding said selected hybridoma cells; and f) isolating the antibodies produced by chromatographic methods.

10. Isolated monoclonal antibodies produced according to the method of claim 9.

11. The isolated monoclonal antibodies of claim 10, wherein said antibodies are IgM.

12. A method for determining the presence of antibodies against the L- asparaginase from Helicobacter pylori CCUG 17874 of claim 6 in a sample, wherein said sample is suspected of containing antibodies against said L-asparaginase, the method comprising the steps of: a) coating an ELISA plate with the L-asparaginase of claim 6; b) contacting the coated ELISA plate with the sample; and c) determining the binding between the antibodies against the L- asparaginase from Helicobacter pylori CCUG 17874 of claim

6 with the L-asparaginase coating provided in step a).

13. The method of claim 12, wherein said sample is a serum sample.

14. The method of claim 12 or 13, wherein said binding is determined spectrophotometrically.

15. The method of any one of claims 12 to 14, wherein said antibodies are IgG and/or IgM.

16. A diagnostic kit comprising an ELISA plate coated with the L- asparaginase of claim 6.

17. L-asparaginase of claim 6 as a medicament.

18. L-asparaginase of claim 6 as an anti-cancer medicament.

19. Use of the L-asparaginase of claim 6 for detecting the presence of antibodies against the L-asparaginase from Helicobacter pylori CCUG 17874 in a serum sample, comprising the step of: a) coating an ELISA plate with the L-asparaginase of claim 6; b) contacting the coated ELISA plate with the serum sample; and c) determining the binding with the L-asparaginase coating provided in step a).

20. Use of the recombinant L-asparaginase from Helicobacter pylori CCUG 17874 of claims 6 as a food additive.

21. A method for reducing the production of acrylamide during the processing of food comprising the steps of: a) applying the L-asparaginase from Helicobacter pylori CCUG 17874 of claim 6 to said food and allowing the enzyme to reduce the amount of asparagine in said food; b) processing said food in order to obtain processed food.

22. The method of claim 21 wherein said food is starch-based food selected from the group comprising potato chips, French fries, potato crisps or croquettes, cereals containing product, wherein said cereals are selected in the group comprising rye, corn, maize, barley, rice and oats; wheat based product; coffee, cocoa, nuts, vegetables and fruits.

23. The method of claim 21 wherein said food processing of step b) includes baking, frying, boiling and roasting.