USE OF GLUTAMINE SYNTHETASE FOR TREATING HYPERAMMONEMIA

Abstract

The present invention relates to the use of glutamine synthetase as a protein therapy (such as enzyme replacement protein therapy) for the treatment of hyperammonemia. In particular the invention relates to the systemic administration of glutamine synthetase. The glutamine synthetase may be provided in conjugated or fusion form, to increase its half-life in the circulation. Also provided is a pharmaceutical composition comprising glutamine synthetase. The invention also relates to the uses, methods, and compositions involving a combination of the glutamine synthetase protein and an ammonia lowering agent, such as a nitrogen scavenger.

Claims

1. A pharmaceutical composition comprising a glutamine synthetase (GS) protein; and a pharmaceutical excipient.

2. The pharmaceutical composition of claim 1, wherein the GS protein is conjugated at the N-terminal end to a peptide comprising a His tag and a linker, and the linker is 2-10 amino acids.

3. The pharmaceutical composition of claim 2, wherein the peptide comprises or consists of the sequence MGSSHHHHHHGGGGS or GSSHHHHHHGGGGS (both SEQ ID NO: 4, in part).

4. The pharmaceutical composition of claim 1 wherein the GS protein is a protein of SEQ ID NO. 1 or a variant thereof having glutamine synthetase enzyme activity and at least 90% sequence identity to the sequence of SEQ ID NO. 1 and/or a fragment thereof having glutamine synthetase enzyme activity and at least 90% of the full length of SEQ ID NO. 1.

5. The pharmaceutical composition of claim 1, wherein the GS protein is conjugated to polyethylene glycol (PEG).

6. The pharmaceutical composition of claim 2 wherein the GS peptide conjugate is conjugated to PEG.

7. The pharmaceutical composition of claim 5, wherein the PEG is linked to the GS at the N terminus.

8. The pharmaceutical composition of claim 6, wherein the PEG is linked to the GS at the N terminus.

9. The pharmaceutical composition of claim 7 wherein the PEG is 20 kDa N-terminal aldehyde PEG.

10. The pharmaceutical composition of claim 8, wherein the PEG is 20 kDa N-terminal aldehyde PEG.

11. The pharmaceutical composition of claim 2, wherein PEG is conjugated to the peptide.

12. The pharmaceutical composition of claim 10, wherein PEG is conjugated to the peptide.

13. The pharmaceutical composition of claim 1, wherein the GS protein is in a form suitable for systemic non-oral administration, and preferably is not administered to muscle.

14. The pharmaceutical composition of claim 1 wherein the GS protein is in a form suitable for parenteral or subcutaneous administration to the subject.

15. The pharmaceutical composition of claim 2, wherein the GS peptide conjugate is in a form suitable for parenteral or subcutaneous administration to the subject.

16. The pharmaceutical composition of claim 1 wherein the GS protein is provided as a preparation comprising multimeric forms of the protein or wherein the GS protein is provided in monomeric form.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0132] The present invention will now be further described with reference to the following non-limiting Examples and Figures in which:

[0133] FIG. 1 shows the results of size exclusion chromatography (SEC) on a Superose 12 column of 20 kDa N-terminal aldehyde PEG conjugates of human GS protein as prepared in Example 1. The graph shows that multimers were eluted in fractions 8 and 9, and monomer in fraction 10;

[0134] FIG. 2 shows a comparison of the in-vitro GS activity of various GS candidates: PEG-conjugated variants (Trin GS1, Trin GS2, Trin GS3, Trin GS 4) versus non-conjugated GS (wt GS) and negative control. Glutamine Synthetase activity is shown as OD 570 nm according to the assay of Acosta et al, 2009, World J. Gastroenterol., 15(23), 2893-2899._Trin GS1—(N-term Ald monomer); Trin GS2—Nof-20; Trin GS3—Nof-30, Trin GS4—N-term Ald multimer;

[0135] FIG. 3 shows PEG ELISA results on plasma pre- and post-dosing of male, wild-type (wt) CD1 mice of various conjugates at (A) baseline, (B) 24 hours post-dose and (C) 72 hours post-dose. (Trin1—N-terminal Aldehyde conjugated GS monomer; Trin2—Nof-20 GS conjugated multimer; Trin 3—Nof-30 conjugated GS multimer; Trin4—N-terminal Aldehyde conjugated PEG multimer);

[0136] FIG. 4 shows GS activity (OD 535 nm) results in liver lysates in dosed wtCD1 mice at 2.5 mg/kg, 3 days post-dosing, as described in Example 3.

[0137] FIG. 5A shows liver GS activity assay results. FIG. 5B shows blood plasma GS activity assay results. Both, liver and blood plasma GS activity was assayed in BDL rats treated with GS protein, and GS protein with nitrogen scavenger.

[0138] FIG. 6 illustrates ammonia blood levels measured in BDL rats treated with GS protein, and GS protein with nitrogen scavenger.

[0139] FIG. 7 illustrates a graph showing percentage of oedema in the prefrontal cortex in BDL rats treated with GS protein, or GS protein with nitrogen scavenger.

[0140] FIG. 8 shows the results of a rotarod grip test in BDL rats treated with GS protein, or GS protein with nitrogen scavenger.

[0141] FIG. 9 a graph showing ammonia levels in OTC mice treated with GS protein, or GS protein with nitrogen scavenger (SP—sodium phenylacetate).

[0142] FIG. 10 shows the results of ammonia levels in plasma and liver GS activity in OTC mice treated with GS protein or DS protein with nitrogen scavenger (SP—sodium phenylacetate)

EXAMPLES

Example 1

Production and Purification of GS Protein and GS Protein-PEG Conjugates

[0143] Production of human glutamine synthetase (GS): pET30a+ vector, containing the gene for human GS (SEQ ID NO: 5 comprising a 5′ sequence encoding a His-tag and the linker GGGGS at the N-terminal end of the GS and codon optimised for expression in bacteria) was used in an E. coli expression system. After plasmid construction, evaluation for the expression of GS was performed with a wide range of induction (IPTG) and expression temperatures. Human GS was solubly expressed in the construct as detected by SDS-PAGE. Lysis buffer (50 mM Tris pH 8.0, 10% glycerol, 0.1% Triton X-100, 100 ug/ml lysozyme, 1 mM PMSF, 3 Units DNAse, 2 mM MgCl) was used to extract soluble protein from cells. Soluble protein was extracted following centrifugation. After expression studies, the best condition found with BL21 (DE3) cells, cultured and induced with 0.1 mM IPTG at 25° C. for 16 hours. Other conditions tried, included using varied IPTG induction (from 0.01M-0.1M IPTG), various incubation temperatures (ranging from 16° C.-37°), and induction incubation times from 4-16 hours.

[0144] Purification of the expressed GS: the first step purification of the expressed protein comprised His tag purification with Ni-NTA beads, washing with 20 mM Imidazole, and elution with 300 mM Imidazole.

[0145] Protein PEG conjugation: the GS protein was conjugated under reducing conditions (with the use of 20 mM Sodium Cyano borohydride) to N-terminal aldehyde 20 kDa peg for 16 hours (Dr Reddy's 20 kDa N-terminal Aldehyde PEG).

[0146] Final purification: Conjugated protein was further purified using SEC chromatography. A Superose 6 or Superose 12 column (see FIG. 1) was used. Multimers were found in fractions 8+9. Fraction 10 in Superose 12 comprised (dilute) multimer. In Superose 6, multimers were found in fractions 8+9 and monomer in fractions 12/13.

[0147] A final formulation of the GS in PBS, pH 7.4 containing trehalose and sucrose was prepared.

Example 2

Activity of GS Preparations

[0148] Various GS preparations and PEG conjugates prepared according to Example 1 were tested for GS activity using the assay of Acosta et al., 2009 (supra), modified from the original assay described in Ehrenfeld et al., 1963, J. Biol. Chem. 238(11), 3711-3716.

[0149] 100 ug of purified protein sample was added to the following reaction buffer: 150 μL stock solution (100 mmol/L imidazole-HCl buffer [pH7.1], 40 mmol/L MgCl2, 50 mmol/L, β-mercaptoethanol, 20 mmol/L ATP, 100 mmol/L, glutamate and 200 mmol/L hydroxylamine, adjusted to pH 7.2) Tubes were incubated at 37° C. for 15 min. The reaction was stopped by adding 0.6 mL [2× concentration] ferric chloride reagent (0.37 mol/L FeCl3, 0.67 mol/L HCl and 0.20 mol/L trichloroacetic acid). Samples were placed for 5 minutes on ice. Precipitated proteins were removed by centrifugation at 10,000 g, and the absorbance of the supernatants was read at 535-570 nm against a reagent blank. The results are shown in FIG. 2. Trin1—(N-term Aldehyde monomer PEG of 20 kD size, obtained from Dr Reddy's); Trin2—Nof-20 conjugated GS, which was conjugated to the GS protein with a monofunctional linear 20 kD PEG, NHS active ester, obtained from NOF corporation); Trin 3—Nof-30, conjugated to the GS protein with a monofunctional linear 30 kD PEG, NHS active ester, obtained from NOF corporation) Trin4—N-term Ald, GS multimers). Trin 4 (N-term Ald multimer) showed the best activity, with a very similar activity profile compared to the wt GS (non-conjugated), though activity of other conjugates was similar.

Example 3

Dosing of GS Protein to Mice—Effects on Plasma Levels of GS Protein-PEG Conjugates

[0150] Male, wild-type (wt) CD1 mice were dosed at 2.5 mg/kg with subcutaneous (s.c) dosing of various GS protein and PEG conjugates prepared as described in Example 1 (Trin1—N-terminal Aldehyde conjugated GS monomer; Trin2—Nof-20 GS conjugated multimer; Trin 3—Nof-30 conjugated GS multimer; Trin4—N-terminal Aldehyde conjugated PEG multimer). The ELISA was conducted according to the protocol outlined by the manufacturer (Abcam PEG ELISA kit, ab133065). Results of the plasma ELISA, as shown in FIG. 3, show either very low or undetectable levels for unconjugated wt GS, as expected at all timepoints. After 24 hours, several candidates were found to be at a high level in plasma; however, after 72 hours post-dosing, Trin-GS 4 (N-terminal Aldehyde conjugated PEG GS multimer) showed the highest presence. N=2 animals in each group. Thus, this experiment shows that systemic administration of GS protein may be used successfully to obtain high circulating levels of the GS PEG conjugates, and in particular at levels which may be therapeutically effective or active.

Example 4

Dosing of GS Protein to Mice—GS Activity Levels of Liver Lysates

[0151] The activity assay was performed as described in Example 2, with the exception that 500 of liver lysate (from culled mice from the experiment of Example 3) was added to each reaction where appropriate. The results are shown in FIG. 4. The GS activity results in liver lysates 3 days post-dosing demonstrate that the superior candidate was the N-terminal aldehyde conjugated PEG GS multimer, which was the only candidate to show significant activity above baseline compared to the vehicle (saline-dosed) control. N=2 animals in each group.

Example 5

[0152] The Otc.sup.spf-ash Mouse model of urea cycle disorder (OTC deficiency) was used to show the effects of GS and GS+SP. The details of the mice used can be found at https://www.jax.org/strain/001811 (B6EiC3Sn a/A-Otc.sup.spf-ash/J). They are fed normal chow. The ages were variable from about 10 weeks to 23 weeks, with groups well-matched. All animals are male hemizygous (as OTC is X-linked, it is present only on the X chromosome of the males, therefore the mice are knockout).

[0153] All groups (vehicle, GS and GS+SP; where GS=Glutamine synthetase, GS+SP=Glutamine Synthetase+Sodium Phenlyacetate) were treated as follows:

[0154] The experiment ran from a Tuesday until the following Wednesday (8 days).

[0155] SP was dosed i.p. 350 mg/kg twice daily; GS was dosed in all treated groups for the first 4 days (i.p. @ 40 mg/kg once daily), then a break of 2 days [a weekend], and 3 more days of dosing with GS @ 40 mg/kg i.p.

[0156] Mice were culled on day 8, and blood extracted, spun down for plasma, and this plasma was used for ammonia quantitation (see method below).

[0157] Genotyping is performed using standard methods described in the literature.

Materials and METHODS

[0158] All experiments were performed in accordance with the Animals (Scientific Procedures) Act of 1986, which was revised according to the European Directive2010/63/EU. All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 86-23; revised 1985). All the animals used in these experiments were Male Sprague-Dawley rats (body weight, 250 g at the beginning of the experiments) were obtained from Charles River Laboratories (Kent, UK) and divided into 5 groups: bile duct ligated animals+ammonia+saline serum (BDL+HA+SS, n=6), bile duct ligated animals+ammonia+sodium phenylacetate (BDL+HA+SP, n=6), bile duct ligated animals+ammonia+sodium phenylacetate+glutamine synthetase (BDL+HA+SP+GS, n=5), bile duct ligated animals+ammonia+glutamine synthetase (BDL+HA+GS, n=6), sham-operated animals+glutamine synthetase (SHAM+GS, n=5). Treatment comprising SP and GS may be referred to as “COMBO”.

Bile Duct Ligation Surgery

[0159] Under general anesthesia (5% isoflurane in 100% oxygen for induction, 2% isofluorane in air for maintenance) rats underwent triple ligation of the bile duct (way of a small laparotomy) to induce chronic liver injury and were studied 28 days after surgery. A midline abdominal incision was made under anesthesia. In the BDL group, the common bile duct was isolated, triply ligated with 3-0 silk, and sectioned between the ligatures. The sham-operated group performed the same procedure without the sectioning between the ligatures. After BDL all animals continued to gain weight and were comparable with sham controls. The overall mortality in both groups was less than 10% and occurred within 36 hours of the operation.

Noncirrhotic Hyperammonemia Condition

[0160] Twenty-three rats were administered a hyperammonemic (HA) diet. The amino acid recipe used for a stock of approximately 100 g was: 15 g leucine, 7.7 g phenylalanine, 7 g glutamate, 10 g alanine, 4.4 g proline, 5.8 g threonine, 11 g aspartate, 5 g serine, 4.8 g glycine, 3.3 g arginine, 9.6 g lysine, 8.4 g histidine, 3 g tyrosine, 1.5 g tryptophan, and 10.6 g valine. 25 g of this mix (mixed 1:5 with standard rodent chow powder) was freshly prepared daily and rats were given free access to it for 5 days. The recipe approximates the amino acid composition of a rodent haemoglobin, [1] mimicking the effect of gastrointestinal bleeding, which is known to result in systemic hyperammonemia [2].

Sodium Phenylacetate Condition

[0161] Eleven rats were administered a sodium phenylacetate (SP) diet. 0.3 g/kg a day for 5 days was mixed with the chow powder and freshly prepared daily.

Glutamine Synthetase Condition

[0162] Sixteen rats were injected with GS intraperitoneally every two days (day 1 and day 3). The total volume injected was 3 mls i.p., which allows for 18-22 mg/kg of GS.

Blood Sampling and Biochemistry

[0163] Plasma samples were collected from the leg vein at different timepoints in all groups. The timepoints were counted after the treatment with glutamine synthetase as follows: 6 hours, 24 hours, 48 hours and 5 days. Analyses were conducted for plasma ammonia levels in every timepoint using 200 μl of respective plasma using a Cobas Integra 400 multi-analyser with the appropriate kits (Roche-diagnostics, Burgess Hill, West Sussex, UK).

Brain Edema

[0164] This was measured using the dry weight technique as described previously [3, 4]. Briefly, oven dried Eppendorf s were weighed with a sensitive electronic scale, then prefrontal cortex, striatum, hippocampus, cerebellum and cortices of each animal were placed into each respectively labelled Eppendorf and reweighed; all samples where within 0.1 mg difference. The dry weight was determined after Eppendorf's loaded with individual brain samples were dried in an oven at 60° C. for 7 days. Tissue water content was then calculated as % H2O=(1-dry wt/wet wt)×100%.

Test for Assessment Locomotor Activity: RotaRod-Accelerod Test

[0165] This test of motor performance consists of a motor-driven rotating rod that enables us to assess motor coordination and resistance to fatigue (Jones and Roberts 1968). The accelerating rotarod 7750 of Ugo Basile (Ugo Basile Biological Research Apparatus, Italy) was used for the rats. The procedure followed has two parts. In the first one, the animals were placed in the apparatus and the speed was maintained constant at 2 rpm for 60 s. In the second part, the rats were evaluated for 5 min in the accelerod test session, in which the rotation rate constantly increased until it reached 20 rpm. Latency to fall off the rod and the actual rotation speed were recorded in the pre- and post-treatment conditions for all groups after 1 hour treatment.

Ammonia Determination in Blood Using the TCA Direct Method

[0166] The method described in the paper (Clin Chim Acta. 1968 October; 22(2)183-86) was used to measure plasma ammonia concentration, as follows.

Principle

[0167] In an alkaline solution ammonium ions react with hypochlorite to form monochloramine. In the presence of phenol and an excess of hypochlorite, the monochloramine will form a blue coloured compound, indophenol, when nitroprusside is used as a catalyst. The concentration of ammonium is determined spectrophotometrically at 630 nm.

Method

[0168] Dissolve 3.5 g of phenol and 0.04 g sodium nitroprusside in 100 ml distilled water to prepare reagent A.

[0169] Dissolve 1.8 g sodium hydroxide in 48 mls in distilled water and add 4 mls of 1M sodium hypochlorite solution to prepare reagent B.

[0170] Add 150 μl of 5% TCA to 50 μl to each plasma sample and centrifuge at 10,000 RPM at 4° c. for 10 minutes. Take 50 μl of the supernatant and put in 96 well plate to which is added 50 μl of both reagents A and B.

[0171] Standard ammonium chloride concentrations for the calibration curve are made by dissolving ammonium chloride in distilled water and serially diluting to make concentrations ranging from 400 μmol to 3 μmol. Distilled water is used as the blank The well plate is covered from light and incubated at 50° c. for 60 minutes. Absorbance is measured at 630 nm using a spectrophotometer to determine the ammonia concentration.

Results

Dosing of GS Protein to Mice—GS Activity Levels in Liver and Blood

[0172] The activity assay was performed as described in the materials and methods section above. The results are shown in FIGS. 5A and B. The results in rat liver measured at day 5, show that GS activity is best in the SHAM+GS group. Additionally, it can been seen from FIG. 5A that GS and GS+SP treatment increase GS activity in the livers of mice which have undergone BDL. When measured in blood, the results show that GS activity is best in the BDL+GS group. Additionally, from FIG. 5B, it can be seen that GS activity in blood is consistant over time, even 24 hrs and 48 hrs post dosing.

Dosing of GS Protein to Mice—Ammonia Concentrations in the BDL Rat

[0173] As seen in FIG. 6, ammonia levels are highest in the BDL rat. Treatment with GS, GS+SP, and SP, each resulted in a significant reduction of ammonia levels in the blood. GS reduced ammonia levels following 2 doses. Treatment with GS+SP reduced the ammonia levels most significantly, suggesting a synergistic effect.

Dosing of GS Protein to Mice—Brain Swelling in the BDL Rat

[0174] Brain oedema was measured in the prefrontal cortex. Treatment with GS was found to reduce brain oedema most significantly compared to treatment to with SP, and even treatment with SP+GC (FIG. 7). Treatment with SP did not statistically significantly reduce the swelling as compared to the control (i.e. BDL mice without treatment).

Dosing of GS Protein to Mice—Brain and Physical Function in the BDL Rat

[0175] FIG. 8 shows the results of a rotarod grip test. Surprisingly, GS dosing was found to improve performance in all tested mice groups. Treatment with SP alone did not lead to statistically significant effects, but treatment with GS+SP shows the best improvement, suggesting a synergistic effect.

Treatment of Mice with OTC Deficiency

[0176] As shown in FIG. 9, ammonia is very significantly decreased in the treated groups.

[0177] In FIG. 10, it is seen that in the OTC mice treated with GS or GS & SP, plasma GS activity increased from 0.2 in the vehicle group, to 0.8 in GS only group and ˜1.1 in the GS & SP group. Liver GS activity increased from 0.175 in the vehicle group, to ˜2.8 in GS only group and ˜2.5 in the GS & SP group

Summary of Results

[0178] In summary, the administered GS is biocompatible, safe and improves blood and liver GS activity. It also leads to a reduction in ammonia and brain oedema, as well as improves neurocognitive and/or physical function. Additionally, the data suggests that treatment with SP and GS may have a synergstic effect.

REFERENCES

[0179] [1] Riggs A. The amino acid composition of some mammalian hemoglobins: mouse, guinea pig, and elephant. J Biol Chem 1963; 238:2983-2987. [0180] [2] Balata S, Olde Damink S W, Ferguson K, Marshall I, Hayes P C, Deutz N E, Williams R, Wardlaw J, Jalan R. Induced hyperammonemia alters neuropsychology, brain MR spectroscopy and magnetization transfer in cirrhosis. Hepatology 2003; 37:931-939. [0181] [3] Stewart-Wallace A M. A biochemical study of cerbral tissue, and of changes in cerebral oedema. Brain 1939; 62: 426-38. [0182] [4] Traber P G, Ganger D R, Blei A T. Brain edema in rabbits with galactosamine-induced fulminant hepatitis. Regional differences and effects on intracranial pressure. Gastroenterology 1986; 91: 1347-56.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

[0183]

TABLE-US-00001 SEQ ID NO: 1 [Full human protein] MTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPEW NFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRI MDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEA HYRACLYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATF DPKPIPGNWNGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNAR RLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRTCLL NETGDEPFQYKN SEQ ID. NO. 2 (ONLY Methionine is cleaved for the mature protein in vivo): TTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPEWNF DGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMD MVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHY RACLYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDP KPIPGNWNGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRL TGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNE TGDEPFQYKN SEQ ID NO: 3 cDNA CGAGAGTGGGAGAAGAGCGGAGCGTGTGAGCAGTACTGCGGCCTCCTCTCCTCTCCTAAC CTGCTCTCGCGGCCTACCTTTACCCGCCCGCCTGCTCGGCGACCAGAACACCTTCCACCA TGACCACCTCAGCAAGTTCCCACTTAAATAAAGGCATCAAGCAGGTGTACATGTCCCTGC CTCAGGGTGAGAAAGTCCAGGCCATGTATATCTGGATCGATGGTACTGGAGAAGGACTGC GCTGCAAGACCCGGACCCTGGACAGTGAGCCCAAGTGTGTGGAAGAGTTGCCTGAGTGGA ATTTCGATGGCTCCAGTACTTTACAGTCTGAGGGTTCCAACAGTGACATGTATCTCGTGC CTGCTGCCATGTTTCGGGACCCCTTCCGTAAGGACCCTAACAAGCTGGTGTTATGTGAAG TTTTCAAGTACAATCGAAGGCCTGCAGAGACCAATTTGAGGCACACCTGTAAACGGATAA TGGACATGGTGAGCAACCAGCACCCCTGGTTTGGCATGGAGCAGGAGTATACCCTCATGG GGACAGATGGGCACCCCTTTGGTTGGCCTTCCAACGGCTTCCCAGGGCCCCAGGGTCCAT ATTACTGTGGTGTGGGAGCAGACAGAGCCTATGGCAGGGACATCGTGGAGGCCCATTACC GGGCCTGCTTGTATGCTGGAGTCAAGATTGCGGGGACTAATGCCGAGGTCATGCCTGCCC AGTGGGAATTTCAGATTGGACCTTGTGAAGGAATCAGCATGGGAGATCATCTCTGGGTGG CCCGTTTCATCTTGCATCGTGTGTGTGAAGACTTTGGAGTGATAGCAACCTTTGATCCTA AGCCCATTCCTGGGAACTGGAATGGTGCAGGCTGCCATACCAACTTCAGCACCAAGGCCA TGCGGGAGGAGAATGGTCTGAAGTACATCGAGGAGGCCATTGAGAAACTAAGCAAGCGGC ACCAGTACCACATCCGTGCCTATGATCCCAAGGGAGGCCTGGACAATGCCCGACGTCTAA CTGGATTCCATGAAACCTCCAACATCAACGACTTTTCTGGTGGTGTAGCCAATCGTAGCG CCAGCATACGCATTCCCCGGACTGTTGGCCAGGAGAAGAAGGGTTACTTTGAAGATCGTC GCCCCTCTGCCAACTGCGACCCCTTTTCGGTGACAGAAGCCCTCATCCGCACGTGTCTTC TCAATGAAACCGGCGATGAGCCCTTCCAGTACAAAAATTAAGTGGACTAGACCTCCAGCT GTTGAGCCCCTCCTAGTTCTTCATCCCACTCCAACTCTTCCCCCTCTCCCAGTTGTCCCG ATTGTAACTCAAAGGGTGGAATATCAAGGTCGTTTTTTTTCATTCC SEQ ID NO: 4: GS protein grown in bacteria, used in Example 1 MGSSHHHHHHGGGGSMTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKT RTLDSEPKCVEELPEWNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKY NRRPAETNLRHTCKRIMDMVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYC GVGADRAYGRDIVEAHYRACLYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVAR FILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYH IRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANC DPFSVTEALIRTCLLNETG DEPFQYKN SEQ ID NO: 5 cDNA (bacterial optimised cDNA used in Example 1). ATGGGCAGCAGCCACCACCATCACCACCACGGCGGCGGCGGTAGCATGACCACCTCGG CAAGCAGCCACCTGAATAAAGGCATCAAACAGGTGTATATGTCTCTGCCGCAGGGTGA AAAAGTTCAAGCCATGTACATTTGGATCGATGGCACCGGTGAAGGCCTGCGTTGCAAA ACCCGCACGCTGGACTCAGAACCGAAATGTGTGGAAGAACTGCCGGAATGGAACTTTG ATGGTAGCTCTACGCTGCAGTCGGAAGGCAGTAATTCCGACATGTATCTGGTTCCGGCG GCCATGTTTCGTGATCCGTTCCGCAAAGACCCGAACAAACTGGTGCTGTGCGAAGTTTT TAAATACAACCGTCGCCCGGCGGAAACCAATCTGCGTCATACGTGTAAACGCATTATG GATATGGTCAGCAACCAGCACCCGTGGTTCGGTATGGAACAAGAATATACCCTGATGG GTACGGATGGCCATCCGTTTGGTTGGCCGAGCAATGGTTTCCCGGGTCCGCAGGGTCCG TATTACTGCGGTGTCGGCGCAGATCGTGCTTACGGTCGCGACATTGTGGAAGCACACTA TCGTGCTTGTCTGTACGCGGGTGTTAAAATCGCCGGCACCAATGCAGAAGTCATGCCG GCTCAGTGGGAATTTCAAATTGGCCCGTGCGAAGGTATCAGCATGGGCGATCATCTGT GGGTTGCTCGTTTCATCCTGCACCGCGTCTGTGAAGATTTTGGTGTGATTGCGACCTTC GACCCGAAACCGATCCCGGGCAACTGGAATGGTGCTGGCTGCCATACCAACTTTAGCA CGAAAGCGATGCGTGAAGAAAATGGCCTGAAATACATCGAAGAAGCAATCGAAAAAC TGTCTAAACGTCATCAGTATCACATTCGCGCCTACGATCCGAAAGGCGGTCTGGACAA CGCACGTCGCCTGACCGGTTTTCACGAAACGAGCAACATCAATGATTTCTCTGCGGGCG TTGCCAATCGCTCAGCCTCGATTCGTATCCCGCGCACCGTCGGTCAAGAGAAAAAAGG CTATTTTGAAGATCGTCGCCCGAGTGCAAACTGTGACCCGTTCTCCGTGACGGAAGCCC TGATCCGCACCTGTCTGCTGAATGAAACCGGCGATGAACCGTTCCAATACAAAAAT SEQ ID NO: 6 [Lactobacillus acidophilus strain 30SC GS] >tr|F0TG87|F0TG87 LACA3 Glutamine synthetase OS = Lactobacillus acidophilus (strain 30SC) MSKQYTTEEIRKEVADKDVRFLRLCFTDINGTEKAVEVPTSQLDKVLTNDIRFDGSSIDGFV RLEESDMVLYPDFSTWSVLPWGDEHGGKIGRLICSVHMTDGKPFAGDPRNNLKRVLGEM KEAGFDTFDIGFEMEFHLFKLDENGNWTTEVPDHASYFDMTSDDEGARCRREIVETLEEIG FEVEAAHHEVGDGQQEIDFRFDDALTTADRCQTFKMVARHIARKHGLFATFMAKPVEGQ AGNGMHNNMSLFKNKHNVFYDKDGEFHLSNTALYFLNGILEHARAITAIGNPTVNSYKRLI PGFEAPVYIAWAAKNRSPLVRIPSAGEINTRLEMRSADPTANPYLLLAACLTAGLKGIKEQK MPMKPVEENIFEMTEEERAEHGIKPLPTTLHNAIKAFKEDDLIKSALGEHLTHSFIESKELEW SKYSQSVSDWERQRYMNW SEO ID NO: 7 [Zea Mays GS] (corn/Maize GS) >tr|B4G1P1|B4G1P1_MAIZE Glutamine synthetase MACLTDLVNLNLSDNTEKIIAEYIWIGGSGMDLRSKARTLSGPVTDPSKLPKWNYDGSSTG QAPGEDSEVILYPQAIFKDPFRRGNNILVMCDCYTPAGEPIPTNKRYNAAKIFSSPEVAAEEP WYGIEQEYTLLQKDTNWPLGWPIGGFPGPQGPYYCGIGAEKSFGRDIVDAHYKACLYAGI NISGINGEVMPGQWEFQVGPSVGISSGDQVWVARYILERITEIAGVVVTFDPKPIPGDWNGA GAHTNYSTESMRKEGGYEVIKAAIEKLKLRHREHIAAYGEGNERRLTGRHETADINTFSWG VANRGASVRVGRETEQNGKGYFEDRRPASNMDPYVVTSMIAETTIIWKP

[0184] In addition to the foregoing, the same Sequence Listings are provided in computer readable form encoded in a file submitted herewith and herein incorporated by reference. The information recorded in computer readable form is identical to the written Sequence Listings provided herein, pursuant to 37 C.F.R. § 1.821(f).