Software Technology for “Method to target cancer treatment and detection using arginine deiminase-fusion protein albumin-binding”

Cannabis Patents and Trademarks – Bing Lou Wong, Norman Fung Man WAI, Sui Yi Kwok, Yun Chung Leung, VISION GLOBAL HOLDINGS Ltd

Abstract for “Method to target cancer treatment and detection using arginine deiminase-fusion protein albumin-binding”

“The invention provides a pharmaceutical composition that contains albumin-binding-arginine deiminase fusion protein (AAD) for the treatment of cancer and other arginine dependent diseases. The AAD fusion proteins can be extracted from both soluble or insoluble crude proteins. They bind to either human serum albumin (HSA), or animal serum albumin. Their high activity and longer half-life allow for efficient depletion in cancer cells. At physiological pH 7.4, the specific activities of AAD fusion protein and wild-type ADI are approximately 20 and 19 U/mg, respectively. To have a synergistic effect in cancer treatment or inhibiting metastasis, the composition can be used either alone or with at least one chemotherapy agent. AAD fusion protein can be used to detect and quantify arginine in various samples, including blood and food.

Background for “Method to target cancer treatment and detection using arginine deiminase-fusion protein albumin-binding”

The incidence of pancreatic, colon, liver, melanoma, and cervical cancers is rising in the world population. These diseases require effective treatments. Many types of cancer, including leukemia and pancreatic, colon, kidney, bladder, lung, prostate and breast cancers, are auxotrophic to arginine. They lack argininosuccinate synetase (ASS), which makes them excellent targets for arginine-depletion therapy.

“Arginine, a semi-essential amino acids for humans and other mammals, is a semi essential amino acid. The urea cycle enzymes ASS and ASL can help to synthesize it from citrulline. The enzyme arginase can convert arginine to ornithine, while ornithine can then be converted to citrulline using the ornithine carbamyltransferase(OTC) found in the mitochondria. You can use the citrulline to make arginine again. Because of the high catalytic activity and abundance of ASS/ASL, normal cells don’t require an extra supply of arginine to grow. Many types of cancers, however, do not have ASS and are therefore auxotrophic to arginine. Their growth depends on the availability of arginine from blood circulation. To inhibit ASS-negative tumor growth, it is possible to target circulating arginine using arginine degrading enzymes [Feun and al., Curr. Pharm. Des. 14:1049-1057 (2008); Kuo et al., Oncotarget. 1:246-251 (2010)].”

Arginase, arginine dcarboxylase and arginine dinase can all degrade arginine. ADI appears to be the most apt for arginine, with a low Km value. ADI converts arginine into citrulline and ammonia. These are the metabolites in the urea cycle. Unfortunately, ADI is not found in prokaryotes, e.g. Mycoplasma species The isolation and purification prokaryotic ADI is not without difficulties. Because of its low enzymatic activity at neutral pH, ADI from Pseudomonas pusida does not show efficacy in vivo. ADI made from Escherichia coli has low enzyme activity and requires multiple denaturation, renaturation processes which increases the production cost.

“As native ADI is found within microorganisms it is antigenic and quickly cleared from patients’ circulation. The native form is immunogenic and can be injected into the bloodstream. It has a short half-life (4 hours) as well as neutralizing antibodies [Ensor et. al., Cancer Res. 62:5443-5450 (2002); Izzo et al., J. Clin. Oncol. 22:1815-1822 (2004)]. Pegylation can correct these shortcomings. ADI bound with PEG via succinimidyl succinate (ADI?PEG 20), is one of the most effective forms of pegylated ADI. The activity of ADI after pegylation decreases by 50% [Ensor et. al., Cancer Res. 62:5443-5450 (2002)]. Pegylated ADI was not possible in the past. This is due to the random attachments of PEG to protein surface Lys residues. It is also difficult to characterize the materials and do quality control during manufacturing. PEG is also very costly, which increases the production cost. Pegylated ADI intravenous in vivo can cause leakage or detachment. The ADI without PEG may also be able to elicit immunogenicity problems. There is an urgent need to improve cancer-treatment compositions.

“In the present invention, albumin binding arginine desiminase (AAD), fusion protein has increased its plasma half-life and activity to efficiently deplete arginase in cancer cells. Native ADI can be found in microorganisms. It is antigenic, fast cleared from the circulation and quickly cleared out of patients’ blood. This invention creates AAD fusion proteins that contain one or two albumin binding proteins. The result is a higher level of activity and a longer in vivo half life (at least five days of arginine loss after one injection). The AAD fusion protein product is not affected by the albumin binding protein. Instead, it appears to increase its circulating half-life. At physiological pH 7.4, the specific enzyme activities of AAD fusion protein and wild-type ADI are approximately 20 and 19 U/mg, respectively. The AAD fusion protein in the present invention is also more soluble than native or wild-type ADI from different organisms. It is generally more difficult to purify native or wild-type ADI than the AAD fusion proteins of the present invention.

The present invention is an albumin binding arginine dminase deiminase protein. It comprises a first section that includes one or more components from an albumin domain, an albumin binding peptide, or an albuminbinding protein, fused to a second part comprising an arginine iminase, to form the albumin bindin arginine fusion protein. This ensures that the albumin bindin arginine fusion protein can also bind to serum albumin

“The invention also relates to pharmaceutical compositions containing albumin-binding-arginine deiminase fusion proteins for targeted cancer treatment in humans or other animals. The present invention focuses on the construction of a modified AAD protein fusion protein that is highly active against cancer cells. The second aspect is to purify AAD-fusion protein with high purity using both insoluble and soluble fractions from crude proteins. AAD fusion protein can bind to albumin in circulation, which will increase its half-life. The third aspect is the invention. The present invention also provides a method for administering the AAD-fusion protein-containing pharmaceutical composition to cancer patients who are suffering from different types of cancers, tumors, or other arginine dependent diseases. AAD fusion protein is used in a test kit to detect arginine.

The AAD fusion protein is modified according to the invention so that it does not dissociate into albumin binding protein and ADI. It also becomes more stable and has a longer circulation half-life. ADI is fused to an albumin-binding domain/peptide/protein in AAD fusion product to extend the plasma half-life and reduce the immunogenicity of the fusion product. The albumin binding domain (ABD), a peptide that binds to albumin in blood, is the fusion product. Different ABD variants can have different or better human serum albumin (HSA), affinity. ABD can be fused to ADI in many different ways. This property, which has a longer half-life, allows for the efficient depletion and removal of arginine in cancerous cells, stem cells, and/or progenitor cells.

The pharmaceutical composition containing AAD Fusion Protein can be used intravenous (i.v.). injection (for quick-acting medication dosage) or intramuscular (i.m.). injection (for a fast-acting, long-lasting dose of medication). The present invention allows for the treatment of many cancers, including pancreatic, liver, brain, and colorectal. The present invention relates to AAD fusion proteins and methods of treating cancer. It also addresses methods for treating and/or inhibiting metastasis from cancerous tissue.

“The present invention allows for the use of a combination of different chemotherapy drugs and/or radiotherapy, as well as the AAD fusion protein to create a synergistic effect in cancer treatment.”

“Arginine, a semi-essential amino acids for humans and other mammals, is a semi essential amino acid. It is made from citrulline in a two-step process that is catalyzed urea cycle enzymes, argininosuccinate synase and argininosuccinate Lyase. The enzyme arginase can convert arginine to ornithine, while ornithine can then be converted to citrulline using the ornithine carbamyltransferase(OTC) found in the mitochondria. You can use the citrulline to make arginine again. Because of the abundance of ASS/ASL’s catalytic activity, normal cells don’t require an extra supply of arginine to grow. Many types of cancers, however, do not have ASS and are thus auxotrophic for AGN. Their growth is dependent solely on arginine from the circulation. To stop ASS-negative tumor growth, it is possible to target circulating arginine using arginine degrading enzymes.

“Arginine deiminase can degrade arginine. ADI converts arginine into citrulline, ammonia and other metabolites of urea. Unfortunately, ADI is not found in prokaryotes, e.g. Mycoplasma species The isolation and purification from prokaryotes of arginine desiminase is not without difficulties. Because of its low enzymatic activity at neutral pH, ADI from Pseudomonas pusta did not show efficacy in vivo. ADI made from Escherichia coli has a low enzymatic activity and requires multiple denaturation, renaturation processes. This increased the production cost. Plasma half-life for the native form is?4 hours after injection into human circulation [Ensor et. al., Cancer Res. 62:5443-5450 (2002); Izzo et al., J. Clin. Oncol. 22:1815-1822 (2004)]. Pegylation can partially correct these shortcomings. ADI bound with PEG via succinimidyl succinate (ADI?PEG 20), is one of the most effective forms of pegylated ADI. The activity of ADI after pegylation is significantly decreased (by?50%) according to Ensor et.al., Cancer Res. 62:5443-5450 (2002); Wang et al., Bioconjug. Chem. 17:1447-1459 (2006)]. The succinimidyl succinate PEG linkinger can be easily hydrolyzed and removed from the protein. This can cause immunogenic problems within a very short time. There is an urgent need for better cancer-treatment compounds, especially those with increased activity.

P. putida ADI was not effective in vivo as it had low enzyme activity at a neutral pH. It was quickly eliminated from the circulation by experimental animals. Takaku et.al. Int. J. J. No. 5,474,928. Antigenicity is a problem in the therapeutic use such a heterologous proteins. Takaku et.al., Jpn. Described the chemical modification of ADI by Mycoplasma arginini via a cyanuric chlorineide linking group with polyethylene glycol. J. Cancer Res., 84(1):1195-1200 (1993). The modification of the protein made it toxic due to the release cyanide from its cyanuric chloride linking groups. However, even for the ADI?PEG20, the PEG linking group can be easily hydrolyzed and removed from the protein. This causes immunogenic problems in a short time. There is therefore a need to create compositions that degrade non-essential amino acid and do not suffer from the same problems as the prior art.

“Many types of cancer, including breast, prostate and colon cancers, as well as liver, lung, kidney, bladder, colon, pancreatic, colon, lymphoma, lung, prostate, renal, liver, and pancreatic cancers, are auxotrophic for arginaine. They lack argininosuccinate synthetase, making them ideal targets for arginine therapy. This invention uses albumin-binding, arginine-deiminase (AAD), fusion proteins that have high activity and long half-lives to efficiently deplete arginine from cancer cells.

“The monomer for ADI has a size of 45 kDa, and it also exists as a dimer (on average of 90 kDa] [Das et.al. Structure. 12:657-667 (2004)]. FIG. 1. One or two albumin-binding domain/peptide/protein(s) with or without linker(s), SEQ ID NO: 46-49, are fused to ADI to form the AAD fusion protein. It is noteworthy that the selection of one or two particular albumin-binding domain/peptide/protein(s) can be made depending upon the type of cancer tissue to be targeted, the desired size and half-life of the resulting fusion protein, and whether a domain or entire protein is selected. The chosen albumin-binding material can be different or the same. A protein and a peptide may be fused together, or two proteins, two domains and a protein can be combined, and the resultant molecule is able to bind serumalbumin without the interference of either the fusion protein or the fusion protein. The position of the albumin-binding domain/peptide/protein is far from the active site. The albumin-binding domain/peptide/protein can be fused to the N-terminus or/and C-terminus of ADI. Different ABD variants can have different or better human serum albumin (HSA), affinity. There are many variants of ABD that can be made and can be fused with ADI. Pseudomonas sp is one example of a micro-organism that has ADI. However, they are not recommended for use due to their potential pathogenicity or pyrogenicity. ADI can come from many microorganisms. Mycoplasma (e.g. Mycoplasma arginini and Mycoplasma arthritidis are Mycoplasma hominis, Mycoplasma arginini and Mycoplasma arthritidis respectively. Lactococcus lactis), Pseudomonas (e.g. Pseudomonas plecoglossicida, Pseudomonas putida, Pseudomonas aeruginosa), Streptococcus (e.g. Streptococcus pyogenes, Streptococcus pneumoniae), Escherichia, Mycobacterium (e.g. Mycobacterium tuberculosis and Bacillus (e.g. Bacillus licheniformis, Bacillus cereus). It is preferable that ADI be cloned using Mycoplasma arginini or Lactococcus Lactis, Bacillus licheniformis Bacillus cereus, Bacillus licheniformis Bacillus cereus, Bacillus licheniformis Bacillus licheniformis or any combination thereof. The sequence alignment of some amino acid sequences shown in FIG. 2 shows their SEQ ID (SEQID NO: 23-35). 2 are also disclosed in this document and in the literatures [Daset al. Structure. 12:657-667 (2004); Wang et al., Bioconjug. Chem. 17:1447-1459 (2006); Ni et al., Appl. Microbiol. Biotechnol. 90:193-201 (2011); El-Sayed et al., Biotechnol Prog. 31(2):396-405 (2015)], in which the disclosures of the literatures is incorporated herein by reference.

FIG. 2 shows the design and sequence of the amino acids for (A), native Mycoplasma argini ADI protein, (B) different AAD fusion Proteins derived from the Mycoplasma argini ADI (SEQID NO: 36-40), and (C) AAD fusion Protein derived from the Bacillus cereus ADI. 3. Different AAD fusion proteins can be successfully made. These embodiments include a linker between the AAD fusion proteins and the albumin-binding Protein.

AAD fusion proteins are easy to produce and purify. FIG. 2 shows how an AAD fusion protein can be successfully expressed in E.coli in both the soluble and insoluble fractions. 8. Furthermore, FIG. FIG. 8 also shows the purified AAD protein that was analyzed using sodium dodecyl-sulfate polyacrylamide electrophoresis. The purified AAD protein fusion protein has a 52.8 kDa size. As shown in FIG. 9).”

“The present invention includes AAD fusion proteins with high activity to deplete arginine from tumor cells for treatment of cancer. The purified AAD fusion proteins has a specific activity that is comparable to the wild-type ADI. MTT assay is used to determine the inhibitory effect of AAD fusion proteins on human cancer cell lines. Different types of cancer cells are seeded on 96-well plates. After 24 hours, they are allowed to acclimatize. After 72 hours, the cells are incubated for 72 hours with AAD at a concentration of 0-10?g/ml. IC50 stands for the half maximal inhibitory dose. It is the amount of AAD fusion proteins required to inhibit 50% of a cancer cell line. The IC50 represents the drug’s effectiveness. The IC50 for AAD fusion protein (amino acids sequence) is shown in FIG. 3E) for various cancer cell lines (human skin cancer, A375 & Sk-mel-28; HCT116; pancreatic cancer in humans, Pancll & Miapaca-2; liver cancer in humans, Sk-hep1 and cervical cancer in humans, C-33A; MDA-MB-231; MDA-MB-231; 22Rv1 for human leukemia, Jurkat).

“TABLE 1\nCancer cell line IC50 of AAD\n(argininosuccinate synthetase-negative, ASS?ve) (?g/ml)\nA375 (human melanoma) 0.104\nSK-mel-28 (human melanoma) 1.92\nPancI (human pancreatic cancer) 0.043\nMia-paca-2 (human pancreatic cancer) 0.010\nSk-hep1 (human liver cancer) >10\nC-33A (human cervical cancer) 0.058\nHCT116 (human colorectal cancer) 0.211\nMDA-MB-231 (human breast cancer) 0.173\n22Rv1 (human prostate cancer) 0.235\nJurkat (human leukemia) 0.379”

“The present invention demonstrated that engineered AAD protein fusion proteins can bind to human serumalbumin (HSA), or any animal serum albumin comparable to HSA. FIG. FIG. 10. This shows that the AAD Fusion Protein (amino Acid Sequence is shown in SEQ ID No: 40, FIG. 3E) is able to bind to HSA easily. The indicated molar ratio HSA is used to incubate AAD for 60 minutes at room temperature. FIG. 10, lanes 1-4. After incubation, the samples are treated with native polyacrylamide gel (10%) Lane 2 shows partial binding of HSA at a ratio of 1:1, while lane 3 shows complete binding at a ratio of 1:5 (HSA-AAD). A mole ratio of 1:1 or 1:5 (i.e. FIG. 10), the formations of the HSA/AAD complex form (?100-110kDa according to the construct in FIG. 1. Using the linker-molecule design. In lane 3, you can clearly see a band of molecular weight?100 kDa that represents the AAD-HSA complexes. (Indicated with an open arrowhead). The formation of the non-covalent HSA/AAD complex is expected to increase the blood’s half-life of AAD protein fusion protein. A long-lasting AAD fusion protein was therefore created.

The AAD fusion protein-containing pharmaceutical formulation of this invention is superior to all other products. The AAD fusion protein-containing pharmaceutical compound of the invention can be used in cancer treatment to reduce the amount of arginine found in tumor tissues. The AAD fusion protein can be combined with other molecular targeting agents or cytotoxic drugs.

“The AAD protein fusion protein of the present invention can be used in a test kit to detect arginine from different samples. The Km value of AAD is small. It indicates that the substrate (arginine) has high affinity for it. The rate will therefore approach the maximum reaction speed more quickly. The AAD fusion protein is useful for testing the arginine levels (1) in cancer patients, (2) in food samples, and (3) in cells.

“EXAMPLES”

“The following examples serve to describe specific embodiments of the invention, but do not limit the invention’s scope.

“Several of these examples relate to making an albumin binding arginine desiminase (fusion protein). There are many techniques that can be used, including cloning or intein-mediated ligation. The term “cloning” is used herein. The term?cloning? is used broadly. It involves creating a fusion genome coding for the albumin binding arginine fusion protein fusion protein, inserting that fusion gene into an vector, inserting it into a host organism, and expressing a protein containing an albumin binding arginine fusion protein. There are many variations of this technique that can be used and they all fall within the scope of the invention.

“Example 1”

“Construction of the Gene Coding for Albumin-Binding Domain/Peptide/Protein (ABD)”

Two rounds of PCR are required to construct the gene code for ABD. The following materials are included in the PCR reaction mix (total volume 25?l).

“50?M mixture of dNTP and phosphorous”

“0.5 Unit of iProofDNAPolmerase (BioRad).”

“10 nM each of these oligos”

“In the second round PCR, the total volume of the PCR mixture is 50?l. It contains the following materials:

“50?M DNTP mixture;

“1?l PCR reactant as DNA Template from the First Round;”

“1 unit of iProofDNAPolmerase (BioRad);”

“200 nM each of these oligos”

“ABD-F7?forward?primer?(SEQ?ID?NO:?07):\n5?-CATGATGCGAATTCCTTAGCTGAAGCTAAAGTCTT\nAGCTAACAGAGAACT-3?\nABD-R8?reverse?primer?(SEQ?ID?NO:?08):\n5?-AGCTACGATAAGCTTAAGGTAATGCAGCTAAAATT\nTCATCTATCAGTG-3?”

“The following PCR programs are used:

Qiagen DNA Gel Extract Kit is able to extract a PCR product that contains the ABD DNA sequence (169 bp), and it can be used for cloning purposes.

“Example 2A”

“Construction and Coding of the Fusion Gene Coding For the AAD Fusion Protein”

“In the first PCR the PCR mixture (total Volume of 50?l) contains these materials:

“50?M DNTP mixture;

“25 ng Mycoplasma arginati genomic DNA;

“1 unit of iProofDNAPolmerase (BioRad);”

“200 nM each of these oligos”

“50?M DNTP mixture;

“10 ng of 1280 bpPCR product;”

“10 ng of 169 bpPCR product;”

“1 unit of iProofDNAPolmerase (BioRad);”

“200 nM each of these oligos”

Qiagen DNA Gel Extract Kit yields a PCR product with 1428 bp. It is then digested using restriction enzymes NdeI, HindIII and ligated with plasmid PREST A (Invitrogen), which has been predigested with these enzymes. The ligation product can then be transformed into E. coli BL21 cells (DE3). DNA sequencing confirms the sequence of the fusion gene.

“Example 2B”

“Cloning His-ABD – PolyN-ADI”

“The construction His-ABD -PolyN ADI (SEQ ID No: 40) in FIG. 3E is accomplished by two steps in overlapping PCR. The PCR fragment from the final step is then inserted into vector pET3a, between the NdeI-BamHI sites. FIG. 6 shows the gene map, nucleotide sequence, and amino acid sequence for His-ABD PolyN-ADI. 6.”

His-ABD-PolyN ADI construction: “Primers in the Construction of His-ABD -PolyN”

“hisABDNde-F?forward?primer?(SEQ?ID?NO:?13):\n5?-GGAGATATACATATGCATCATCACCATCACCATGATGAAG\nCCGTGGATG-3?\nABDnn-R1?reverse?primer?(SEQ?ID?NO:?14):\n5?-TTGTTATTATTGTTGTTACTACCCGAAGGTAATGCAGCTA\nAAATTTCATC-3?\nABDn-R2?reverse?primer?(SEQ?ID?NO:?15):\n5?-AGAACCGCCGCTACCATTGTTATTATTGTTGTTACTACCC\nGA-3?\nADln-F?forward?primer?(SEQ?ID?NO:?16):\n5?-AATAATAACAATGGTAGCGGCGGTTCTGTATTTGACAGTA\nAATTTAAAGG-3?\nADIBam-R?reverse?primer?(SEQ?ID?NO:?17):\n5?-TAGATCAATGGATCCTTACCACTTAACATCTTTACGTGAT\nAAAG-3?”

“In the first round PCR, 50 ml of the reaction volume containing known components is prepared in two PCR tubes. Each tube contains dNTP (BIO?RAD), iProof buffer(BIO?RAD), iProofDNA polymerase (BIO?RAD), primers, and the DNA template. The tubes are then mixed with ddH2O to make 50?l. The DNA template is a pET3a Vector containing the gene for ADI from Mycoplasma arginini. It has an internal NdeI site mutation removed without altering its protein sequence.

“The primer mixtures for (A) 10 mg hisABDNdeF (SEQID NO: 13), (0.5 pmol) ABDnn?R1 (SEQID NO: 14), and (10 pmol) ABDn?R2 (SEQID NO: 15); (B) 10 and 10 respectively pmols ADIn?F (SEQID NO: 16), and (Pmol ADIBam?R (SEQID NO: 17), respectively.

The second overlapping step prepares the reaction mixture in the same way as the first round, except that the template was 1 mol of the 237-bp PCR product and 1 mole of the 1278-bp product from the first round of PCR. The primers used have been changed to 10 mg hisABDNdeF (SEQID NO: 13), and 10 mg ADIBamR (SEQID NO: 17).

“Example 2C”

“Cloning His-ABD – PolyN-bcADI

“The construction His-ABD -PolyN -bcADI (SEQ Id NO: 41) in FIG. 3F is achieved by two steps in overlapping PCR. The PCR fragment from the last step is then inserted into vector pET3a, between the NdeI-BamHI sites. FIG. 7 shows the gene map, nucleotide sequence, and amino acid sequence for His-ABD-PolyN -bcADI. 7.”

“Primers involved in construction of His-ABD-PolyN-bcADI:”

“hisABDNde-F2?forward?primer?(SEQ?ID?NO:?18):\n5?-GGAGATATACATATGCATCATCACCATCACCATGATGAAGC\nCGTGGATG-3?\nbcABDnn-R1?reverse?primer?(SEQ?ID?NO:?19):\n5?-TTGTTATTATTGTTGTTACTACCCGAAGGTAATGCAGCTAA\nAATTTCATC-3?\nbcABDn-R2?reverse?primer?(SEQ?ID?NO:?20):\n5?-TTTACCGCCGCTACCATTGTTATTATTGTTGTTACTACCCG\nA-3?\nbcADln-F?forward?primer?(SEQ?ID?NO:?21):\n5?-AATAATAACAATGGTAGCGGCGGTAAACATCCGATACATGT\nTACTTCAGA-3?\nbcADIBam-R?reverse?primer?(SEQ?ID?NO:?22):\n5?-TAGATCAATGGATCCCTAAATATCTTTACGAACAATTGGCA\nTAC-3?”

“The first round of PCR involves 50?l reaction volume with the known concentrations of components. This is done in two PCR tubes. Each tube contains dNTP (BIO?RAD), iProof buffer(BIO?RAD), iProofDNA polymerase (BIO?RAD), primers, and the DNA template. The tubes are then mixed with ddH2O to make 50?l. The DNA template is a pET3a Vector containing the gene for ADI from Bacillius cereus. It has an internal NdeI site mutation removed without altering its protein sequence.

The second overlapping step prepares the reaction mixture in the same way as the first round, except that the template is 1 mol of the 237-bp PCR product and 1 mole of the 1250-bp product from the first round of PCR. The primers used have been changed to 10 mg hisABDNdeF2 (SEQID NO: 18), and 10 mg bcADIBamR (SEQID NO: 22).

“Example 3”

“Expression of the AAD Fusion Protein and its Purification”

“(3a). Expression of AAD Fusion Protein using Shake-Flask Method

“(3b). Expression of AAD Fusion Protein By Fermentation Method”

“(3c). Purification of AAD Fusion Protein”

After centrifugation, the soluble portion of the protein is collected. The nickel affinity chromatography is used to purify the fusion protein, which contains a His tag. TABLE 2 shows how the cultivation temperature affects the solubility AAD fusion proteins (amino acids sequence is shown in FIG. SEQ ID NO. 40, FIG. 3E) is obtained from the expression host.

The cell pellet should be resuspended in 25ml of 10mM sodiumphosphate buffer pH 7.4. The cells can be lysed using sonication and/or a high pressure homogenizer. After centrifugation, the soluble portion of the cells is separated. After centrifugation, the AAD fusion protein (with or without a His tag) can be purified using nickel affinity chromatography and/or Ion-Exchange columns.

The cell pellet can be used to isolate the insoluble fraction AAD fusion proteins. It is resuspended with 25 ml 20 mM TrisHCl, pH 7.4, 1% TRITON X100. The cells are lysed using sonication. Centrifugation is used to collect the insoluble portions (inclusion bodies). After the protein has been dissolved, it is resuspended in 10 ml 20 mM TrisHCl, pH 7.4, 6 m Guanidine HCl and vortexed to make it soluble. You can refold the protein by dropping it into 100 ml (20 mM) Sodium phosphate buffer pH 7.4. Centrifugation is used to remove insoluble substances. To achieve 70% saturation, salting the protein can be done by adding solid ammonium sulfurate powder to the supernatant. Centrifugation separates the insoluble part and it is then resuspended with 10 ml 20 mM sodiumphosphate buffer. The AAD fusion protein, which may contain a His tag (or not), is then purified using nickel affinity chromatography or ion-exchange columns.

TABLE 3 shows the yield and enzyme activity of AAD Fusion Protein from Shake-Flask Method and Fermentation Method.

“TABLE 3\nActivity\nAAD Yield (mg/L) (U/mg)\nShake-flask method ~10 ~9\nFermentation method ~42 ~19”

“Example 4”

“Enzyme Activity Assay & Enzyme Kinetics For AAD Fusion Protein”

“To determine the enzyme activity for wild-type ADI and AAD fusion protein in the present invention, the diacetyl monoxime (DAM)-thiosemicarbazide (TSC) assay for citrulline detection is used. The reaction is shown below.\nL-Arginine\nargininedeiminase(ADI)orAADfusion >L-Citrulline+Ammonia”

The Michaelis constant Km, which is an inverted measure of the substrate’s affinity to the enzyme, is the concentration at which reaction rate is at half-maximum. Km below 0.5 indicates a high affinity for the substrate. This means that the reaction rate will reach the maximum rate faster. The enzyme kinetics, or Km, of wild-type ADI (or AAD fusion protein) are determined under various concentrations of substrate arginine (2000??M, 1000??M, 500???M, 250??M), at pH 7.4. FIG. 3E shows the Km values for AAD fusion proteins. 3E (SEQID NO: 40), ADI protein is derived from Mycoplasma arginini. FIG. 3F (SEQID NO: 41), ADI protein is from Bacillus cereus), are 0.0041 mM, 0.132 mM, respectively. These results indicate that ABD fusion did not alter the binding affinity of different AAD fusion proteins for arginine.

“Example 5”

Summary for “Method to target cancer treatment and detection using arginine deiminase-fusion protein albumin-binding”

The incidence of pancreatic, colon, liver, melanoma, and cervical cancers is rising in the world population. These diseases require effective treatments. Many types of cancer, including leukemia and pancreatic, colon, kidney, bladder, lung, prostate and breast cancers, are auxotrophic to arginine. They lack argininosuccinate synetase (ASS), which makes them excellent targets for arginine-depletion therapy.

“Arginine, a semi-essential amino acids for humans and other mammals, is a semi essential amino acid. The urea cycle enzymes ASS and ASL can help to synthesize it from citrulline. The enzyme arginase can convert arginine to ornithine, while ornithine can then be converted to citrulline using the ornithine carbamyltransferase(OTC) found in the mitochondria. You can use the citrulline to make arginine again. Because of the high catalytic activity and abundance of ASS/ASL, normal cells don’t require an extra supply of arginine to grow. Many types of cancers, however, do not have ASS and are therefore auxotrophic to arginine. Their growth depends on the availability of arginine from blood circulation. To inhibit ASS-negative tumor growth, it is possible to target circulating arginine using arginine degrading enzymes [Feun and al., Curr. Pharm. Des. 14:1049-1057 (2008); Kuo et al., Oncotarget. 1:246-251 (2010)].”

Arginase, arginine dcarboxylase and arginine dinase can all degrade arginine. ADI appears to be the most apt for arginine, with a low Km value. ADI converts arginine into citrulline and ammonia. These are the metabolites in the urea cycle. Unfortunately, ADI is not found in prokaryotes, e.g. Mycoplasma species The isolation and purification prokaryotic ADI is not without difficulties. Because of its low enzymatic activity at neutral pH, ADI from Pseudomonas pusida does not show efficacy in vivo. ADI made from Escherichia coli has low enzyme activity and requires multiple denaturation, renaturation processes which increases the production cost.

“As native ADI is found within microorganisms it is antigenic and quickly cleared from patients’ circulation. The native form is immunogenic and can be injected into the bloodstream. It has a short half-life (4 hours) as well as neutralizing antibodies [Ensor et. al., Cancer Res. 62:5443-5450 (2002); Izzo et al., J. Clin. Oncol. 22:1815-1822 (2004)]. Pegylation can correct these shortcomings. ADI bound with PEG via succinimidyl succinate (ADI?PEG 20), is one of the most effective forms of pegylated ADI. The activity of ADI after pegylation decreases by 50% [Ensor et. al., Cancer Res. 62:5443-5450 (2002)]. Pegylated ADI was not possible in the past. This is due to the random attachments of PEG to protein surface Lys residues. It is also difficult to characterize the materials and do quality control during manufacturing. PEG is also very costly, which increases the production cost. Pegylated ADI intravenous in vivo can cause leakage or detachment. The ADI without PEG may also be able to elicit immunogenicity problems. There is an urgent need to improve cancer-treatment compositions.

“In the present invention, albumin binding arginine desiminase (AAD), fusion protein has increased its plasma half-life and activity to efficiently deplete arginase in cancer cells. Native ADI can be found in microorganisms. It is antigenic, fast cleared from the circulation and quickly cleared out of patients’ blood. This invention creates AAD fusion proteins that contain one or two albumin binding proteins. The result is a higher level of activity and a longer in vivo half life (at least five days of arginine loss after one injection). The AAD fusion protein product is not affected by the albumin binding protein. Instead, it appears to increase its circulating half-life. At physiological pH 7.4, the specific enzyme activities of AAD fusion protein and wild-type ADI are approximately 20 and 19 U/mg, respectively. The AAD fusion protein in the present invention is also more soluble than native or wild-type ADI from different organisms. It is generally more difficult to purify native or wild-type ADI than the AAD fusion proteins of the present invention.

The present invention is an albumin binding arginine dminase deiminase protein. It comprises a first section that includes one or more components from an albumin domain, an albumin binding peptide, or an albuminbinding protein, fused to a second part comprising an arginine iminase, to form the albumin bindin arginine fusion protein. This ensures that the albumin bindin arginine fusion protein can also bind to serum albumin

“The invention also relates to pharmaceutical compositions containing albumin-binding-arginine deiminase fusion proteins for targeted cancer treatment in humans or other animals. The present invention focuses on the construction of a modified AAD protein fusion protein that is highly active against cancer cells. The second aspect is to purify AAD-fusion protein with high purity using both insoluble and soluble fractions from crude proteins. AAD fusion protein can bind to albumin in circulation, which will increase its half-life. The third aspect is the invention. The present invention also provides a method for administering the AAD-fusion protein-containing pharmaceutical composition to cancer patients who are suffering from different types of cancers, tumors, or other arginine dependent diseases. AAD fusion protein is used in a test kit to detect arginine.

The AAD fusion protein is modified according to the invention so that it does not dissociate into albumin binding protein and ADI. It also becomes more stable and has a longer circulation half-life. ADI is fused to an albumin-binding domain/peptide/protein in AAD fusion product to extend the plasma half-life and reduce the immunogenicity of the fusion product. The albumin binding domain (ABD), a peptide that binds to albumin in blood, is the fusion product. Different ABD variants can have different or better human serum albumin (HSA), affinity. ABD can be fused to ADI in many different ways. This property, which has a longer half-life, allows for the efficient depletion and removal of arginine in cancerous cells, stem cells, and/or progenitor cells.

The pharmaceutical composition containing AAD Fusion Protein can be used intravenous (i.v.). injection (for quick-acting medication dosage) or intramuscular (i.m.). injection (for a fast-acting, long-lasting dose of medication). The present invention allows for the treatment of many cancers, including pancreatic, liver, brain, and colorectal. The present invention relates to AAD fusion proteins and methods of treating cancer. It also addresses methods for treating and/or inhibiting metastasis from cancerous tissue.

“The present invention allows for the use of a combination of different chemotherapy drugs and/or radiotherapy, as well as the AAD fusion protein to create a synergistic effect in cancer treatment.”

“Arginine, a semi-essential amino acids for humans and other mammals, is a semi essential amino acid. It is made from citrulline in a two-step process that is catalyzed urea cycle enzymes, argininosuccinate synase and argininosuccinate Lyase. The enzyme arginase can convert arginine to ornithine, while ornithine can then be converted to citrulline using the ornithine carbamyltransferase(OTC) found in the mitochondria. You can use the citrulline to make arginine again. Because of the abundance of ASS/ASL’s catalytic activity, normal cells don’t require an extra supply of arginine to grow. Many types of cancers, however, do not have ASS and are thus auxotrophic for AGN. Their growth is dependent solely on arginine from the circulation. To stop ASS-negative tumor growth, it is possible to target circulating arginine using arginine degrading enzymes.

“Arginine deiminase can degrade arginine. ADI converts arginine into citrulline, ammonia and other metabolites of urea. Unfortunately, ADI is not found in prokaryotes, e.g. Mycoplasma species The isolation and purification from prokaryotes of arginine desiminase is not without difficulties. Because of its low enzymatic activity at neutral pH, ADI from Pseudomonas pusta did not show efficacy in vivo. ADI made from Escherichia coli has a low enzymatic activity and requires multiple denaturation, renaturation processes. This increased the production cost. Plasma half-life for the native form is?4 hours after injection into human circulation [Ensor et. al., Cancer Res. 62:5443-5450 (2002); Izzo et al., J. Clin. Oncol. 22:1815-1822 (2004)]. Pegylation can partially correct these shortcomings. ADI bound with PEG via succinimidyl succinate (ADI?PEG 20), is one of the most effective forms of pegylated ADI. The activity of ADI after pegylation is significantly decreased (by?50%) according to Ensor et.al., Cancer Res. 62:5443-5450 (2002); Wang et al., Bioconjug. Chem. 17:1447-1459 (2006)]. The succinimidyl succinate PEG linkinger can be easily hydrolyzed and removed from the protein. This can cause immunogenic problems within a very short time. There is an urgent need for better cancer-treatment compounds, especially those with increased activity.

P. putida ADI was not effective in vivo as it had low enzyme activity at a neutral pH. It was quickly eliminated from the circulation by experimental animals. Takaku et.al. Int. J. J. No. 5,474,928. Antigenicity is a problem in the therapeutic use such a heterologous proteins. Takaku et.al., Jpn. Described the chemical modification of ADI by Mycoplasma arginini via a cyanuric chlorineide linking group with polyethylene glycol. J. Cancer Res., 84(1):1195-1200 (1993). The modification of the protein made it toxic due to the release cyanide from its cyanuric chloride linking groups. However, even for the ADI?PEG20, the PEG linking group can be easily hydrolyzed and removed from the protein. This causes immunogenic problems in a short time. There is therefore a need to create compositions that degrade non-essential amino acid and do not suffer from the same problems as the prior art.

“Many types of cancer, including breast, prostate and colon cancers, as well as liver, lung, kidney, bladder, colon, pancreatic, colon, lymphoma, lung, prostate, renal, liver, and pancreatic cancers, are auxotrophic for arginaine. They lack argininosuccinate synthetase, making them ideal targets for arginine therapy. This invention uses albumin-binding, arginine-deiminase (AAD), fusion proteins that have high activity and long half-lives to efficiently deplete arginine from cancer cells.

“The monomer for ADI has a size of 45 kDa, and it also exists as a dimer (on average of 90 kDa] [Das et.al. Structure. 12:657-667 (2004)]. FIG. 1. One or two albumin-binding domain/peptide/protein(s) with or without linker(s), SEQ ID NO: 46-49, are fused to ADI to form the AAD fusion protein. It is noteworthy that the selection of one or two particular albumin-binding domain/peptide/protein(s) can be made depending upon the type of cancer tissue to be targeted, the desired size and half-life of the resulting fusion protein, and whether a domain or entire protein is selected. The chosen albumin-binding material can be different or the same. A protein and a peptide may be fused together, or two proteins, two domains and a protein can be combined, and the resultant molecule is able to bind serumalbumin without the interference of either the fusion protein or the fusion protein. The position of the albumin-binding domain/peptide/protein is far from the active site. The albumin-binding domain/peptide/protein can be fused to the N-terminus or/and C-terminus of ADI. Different ABD variants can have different or better human serum albumin (HSA), affinity. There are many variants of ABD that can be made and can be fused with ADI. Pseudomonas sp is one example of a micro-organism that has ADI. However, they are not recommended for use due to their potential pathogenicity or pyrogenicity. ADI can come from many microorganisms. Mycoplasma (e.g. Mycoplasma arginini and Mycoplasma arthritidis are Mycoplasma hominis, Mycoplasma arginini and Mycoplasma arthritidis respectively. Lactococcus lactis), Pseudomonas (e.g. Pseudomonas plecoglossicida, Pseudomonas putida, Pseudomonas aeruginosa), Streptococcus (e.g. Streptococcus pyogenes, Streptococcus pneumoniae), Escherichia, Mycobacterium (e.g. Mycobacterium tuberculosis and Bacillus (e.g. Bacillus licheniformis, Bacillus cereus). It is preferable that ADI be cloned using Mycoplasma arginini or Lactococcus Lactis, Bacillus licheniformis Bacillus cereus, Bacillus licheniformis Bacillus cereus, Bacillus licheniformis Bacillus licheniformis or any combination thereof. The sequence alignment of some amino acid sequences shown in FIG. 2 shows their SEQ ID (SEQID NO: 23-35). 2 are also disclosed in this document and in the literatures [Daset al. Structure. 12:657-667 (2004); Wang et al., Bioconjug. Chem. 17:1447-1459 (2006); Ni et al., Appl. Microbiol. Biotechnol. 90:193-201 (2011); El-Sayed et al., Biotechnol Prog. 31(2):396-405 (2015)], in which the disclosures of the literatures is incorporated herein by reference.

FIG. 2 shows the design and sequence of the amino acids for (A), native Mycoplasma argini ADI protein, (B) different AAD fusion Proteins derived from the Mycoplasma argini ADI (SEQID NO: 36-40), and (C) AAD fusion Protein derived from the Bacillus cereus ADI. 3. Different AAD fusion proteins can be successfully made. These embodiments include a linker between the AAD fusion proteins and the albumin-binding Protein.

AAD fusion proteins are easy to produce and purify. FIG. 2 shows how an AAD fusion protein can be successfully expressed in E.coli in both the soluble and insoluble fractions. 8. Furthermore, FIG. FIG. 8 also shows the purified AAD protein that was analyzed using sodium dodecyl-sulfate polyacrylamide electrophoresis. The purified AAD protein fusion protein has a 52.8 kDa size. As shown in FIG. 9).”

“The present invention includes AAD fusion proteins with high activity to deplete arginine from tumor cells for treatment of cancer. The purified AAD fusion proteins has a specific activity that is comparable to the wild-type ADI. MTT assay is used to determine the inhibitory effect of AAD fusion proteins on human cancer cell lines. Different types of cancer cells are seeded on 96-well plates. After 24 hours, they are allowed to acclimatize. After 72 hours, the cells are incubated for 72 hours with AAD at a concentration of 0-10?g/ml. IC50 stands for the half maximal inhibitory dose. It is the amount of AAD fusion proteins required to inhibit 50% of a cancer cell line. The IC50 represents the drug’s effectiveness. The IC50 for AAD fusion protein (amino acids sequence) is shown in FIG. 3E) for various cancer cell lines (human skin cancer, A375 & Sk-mel-28; HCT116; pancreatic cancer in humans, Pancll & Miapaca-2; liver cancer in humans, Sk-hep1 and cervical cancer in humans, C-33A; MDA-MB-231; MDA-MB-231; 22Rv1 for human leukemia, Jurkat).

“TABLE 1\nCancer cell line IC50 of AAD\n(argininosuccinate synthetase-negative, ASS?ve) (?g/ml)\nA375 (human melanoma) 0.104\nSK-mel-28 (human melanoma) 1.92\nPancI (human pancreatic cancer) 0.043\nMia-paca-2 (human pancreatic cancer) 0.010\nSk-hep1 (human liver cancer) >10\nC-33A (human cervical cancer) 0.058\nHCT116 (human colorectal cancer) 0.211\nMDA-MB-231 (human breast cancer) 0.173\n22Rv1 (human prostate cancer) 0.235\nJurkat (human leukemia) 0.379”

“The present invention demonstrated that engineered AAD protein fusion proteins can bind to human serumalbumin (HSA), or any animal serum albumin comparable to HSA. FIG. FIG. 10. This shows that the AAD Fusion Protein (amino Acid Sequence is shown in SEQ ID No: 40, FIG. 3E) is able to bind to HSA easily. The indicated molar ratio HSA is used to incubate AAD for 60 minutes at room temperature. FIG. 10, lanes 1-4. After incubation, the samples are treated with native polyacrylamide gel (10%) Lane 2 shows partial binding of HSA at a ratio of 1:1, while lane 3 shows complete binding at a ratio of 1:5 (HSA-AAD). A mole ratio of 1:1 or 1:5 (i.e. FIG. 10), the formations of the HSA/AAD complex form (?100-110kDa according to the construct in FIG. 1. Using the linker-molecule design. In lane 3, you can clearly see a band of molecular weight?100 kDa that represents the AAD-HSA complexes. (Indicated with an open arrowhead). The formation of the non-covalent HSA/AAD complex is expected to increase the blood’s half-life of AAD protein fusion protein. A long-lasting AAD fusion protein was therefore created.

The AAD fusion protein-containing pharmaceutical formulation of this invention is superior to all other products. The AAD fusion protein-containing pharmaceutical compound of the invention can be used in cancer treatment to reduce the amount of arginine found in tumor tissues. The AAD fusion protein can be combined with other molecular targeting agents or cytotoxic drugs.

“The AAD protein fusion protein of the present invention can be used in a test kit to detect arginine from different samples. The Km value of AAD is small. It indicates that the substrate (arginine) has high affinity for it. The rate will therefore approach the maximum reaction speed more quickly. The AAD fusion protein is useful for testing the arginine levels (1) in cancer patients, (2) in food samples, and (3) in cells.

“EXAMPLES”

“The following examples serve to describe specific embodiments of the invention, but do not limit the invention’s scope.

“Several of these examples relate to making an albumin binding arginine desiminase (fusion protein). There are many techniques that can be used, including cloning or intein-mediated ligation. The term “cloning” is used herein. The term?cloning? is used broadly. It involves creating a fusion genome coding for the albumin binding arginine fusion protein fusion protein, inserting that fusion gene into an vector, inserting it into a host organism, and expressing a protein containing an albumin binding arginine fusion protein. There are many variations of this technique that can be used and they all fall within the scope of the invention.

“Example 1”

“Construction of the Gene Coding for Albumin-Binding Domain/Peptide/Protein (ABD)”

Two rounds of PCR are required to construct the gene code for ABD. The following materials are included in the PCR reaction mix (total volume 25?l).

“50?M mixture of dNTP and phosphorous”

“0.5 Unit of iProofDNAPolmerase (BioRad).”

“10 nM each of these oligos”

“In the second round PCR, the total volume of the PCR mixture is 50?l. It contains the following materials:

“50?M DNTP mixture;

“1?l PCR reactant as DNA Template from the First Round;”

“1 unit of iProofDNAPolmerase (BioRad);”

“200 nM each of these oligos”

“ABD-F7?forward?primer?(SEQ?ID?NO:?07):\n5?-CATGATGCGAATTCCTTAGCTGAAGCTAAAGTCTT\nAGCTAACAGAGAACT-3?\nABD-R8?reverse?primer?(SEQ?ID?NO:?08):\n5?-AGCTACGATAAGCTTAAGGTAATGCAGCTAAAATT\nTCATCTATCAGTG-3?”

“The following PCR programs are used:

Qiagen DNA Gel Extract Kit is able to extract a PCR product that contains the ABD DNA sequence (169 bp), and it can be used for cloning purposes.

“Example 2A”

“Construction and Coding of the Fusion Gene Coding For the AAD Fusion Protein”

“In the first PCR the PCR mixture (total Volume of 50?l) contains these materials:

“50?M DNTP mixture;

“25 ng Mycoplasma arginati genomic DNA;

“1 unit of iProofDNAPolmerase (BioRad);”

“200 nM each of these oligos”

“50?M DNTP mixture;

“10 ng of 1280 bpPCR product;”

“10 ng of 169 bpPCR product;”

“1 unit of iProofDNAPolmerase (BioRad);”

“200 nM each of these oligos”

Qiagen DNA Gel Extract Kit yields a PCR product with 1428 bp. It is then digested using restriction enzymes NdeI, HindIII and ligated with plasmid PREST A (Invitrogen), which has been predigested with these enzymes. The ligation product can then be transformed into E. coli BL21 cells (DE3). DNA sequencing confirms the sequence of the fusion gene.

“Example 2B”

“Cloning His-ABD – PolyN-ADI”

“The construction His-ABD -PolyN ADI (SEQ ID No: 40) in FIG. 3E is accomplished by two steps in overlapping PCR. The PCR fragment from the final step is then inserted into vector pET3a, between the NdeI-BamHI sites. FIG. 6 shows the gene map, nucleotide sequence, and amino acid sequence for His-ABD PolyN-ADI. 6.”

His-ABD-PolyN ADI construction: “Primers in the Construction of His-ABD -PolyN”

“hisABDNde-F?forward?primer?(SEQ?ID?NO:?13):\n5?-GGAGATATACATATGCATCATCACCATCACCATGATGAAG\nCCGTGGATG-3?\nABDnn-R1?reverse?primer?(SEQ?ID?NO:?14):\n5?-TTGTTATTATTGTTGTTACTACCCGAAGGTAATGCAGCTA\nAAATTTCATC-3?\nABDn-R2?reverse?primer?(SEQ?ID?NO:?15):\n5?-AGAACCGCCGCTACCATTGTTATTATTGTTGTTACTACCC\nGA-3?\nADln-F?forward?primer?(SEQ?ID?NO:?16):\n5?-AATAATAACAATGGTAGCGGCGGTTCTGTATTTGACAGTA\nAATTTAAAGG-3?\nADIBam-R?reverse?primer?(SEQ?ID?NO:?17):\n5?-TAGATCAATGGATCCTTACCACTTAACATCTTTACGTGAT\nAAAG-3?”

“In the first round PCR, 50 ml of the reaction volume containing known components is prepared in two PCR tubes. Each tube contains dNTP (BIO?RAD), iProof buffer(BIO?RAD), iProofDNA polymerase (BIO?RAD), primers, and the DNA template. The tubes are then mixed with ddH2O to make 50?l. The DNA template is a pET3a Vector containing the gene for ADI from Mycoplasma arginini. It has an internal NdeI site mutation removed without altering its protein sequence.

“The primer mixtures for (A) 10 mg hisABDNdeF (SEQID NO: 13), (0.5 pmol) ABDnn?R1 (SEQID NO: 14), and (10 pmol) ABDn?R2 (SEQID NO: 15); (B) 10 and 10 respectively pmols ADIn?F (SEQID NO: 16), and (Pmol ADIBam?R (SEQID NO: 17), respectively.

The second overlapping step prepares the reaction mixture in the same way as the first round, except that the template was 1 mol of the 237-bp PCR product and 1 mole of the 1278-bp product from the first round of PCR. The primers used have been changed to 10 mg hisABDNdeF (SEQID NO: 13), and 10 mg ADIBamR (SEQID NO: 17).

“Example 2C”

“Cloning His-ABD – PolyN-bcADI

“The construction His-ABD -PolyN -bcADI (SEQ Id NO: 41) in FIG. 3F is achieved by two steps in overlapping PCR. The PCR fragment from the last step is then inserted into vector pET3a, between the NdeI-BamHI sites. FIG. 7 shows the gene map, nucleotide sequence, and amino acid sequence for His-ABD-PolyN -bcADI. 7.”

“Primers involved in construction of His-ABD-PolyN-bcADI:”

“hisABDNde-F2?forward?primer?(SEQ?ID?NO:?18):\n5?-GGAGATATACATATGCATCATCACCATCACCATGATGAAGC\nCGTGGATG-3?\nbcABDnn-R1?reverse?primer?(SEQ?ID?NO:?19):\n5?-TTGTTATTATTGTTGTTACTACCCGAAGGTAATGCAGCTAA\nAATTTCATC-3?\nbcABDn-R2?reverse?primer?(SEQ?ID?NO:?20):\n5?-TTTACCGCCGCTACCATTGTTATTATTGTTGTTACTACCCG\nA-3?\nbcADln-F?forward?primer?(SEQ?ID?NO:?21):\n5?-AATAATAACAATGGTAGCGGCGGTAAACATCCGATACATGT\nTACTTCAGA-3?\nbcADIBam-R?reverse?primer?(SEQ?ID?NO:?22):\n5?-TAGATCAATGGATCCCTAAATATCTTTACGAACAATTGGCA\nTAC-3?”

“The first round of PCR involves 50?l reaction volume with the known concentrations of components. This is done in two PCR tubes. Each tube contains dNTP (BIO?RAD), iProof buffer(BIO?RAD), iProofDNA polymerase (BIO?RAD), primers, and the DNA template. The tubes are then mixed with ddH2O to make 50?l. The DNA template is a pET3a Vector containing the gene for ADI from Bacillius cereus. It has an internal NdeI site mutation removed without altering its protein sequence.

The second overlapping step prepares the reaction mixture in the same way as the first round, except that the template is 1 mol of the 237-bp PCR product and 1 mole of the 1250-bp product from the first round of PCR. The primers used have been changed to 10 mg hisABDNdeF2 (SEQID NO: 18), and 10 mg bcADIBamR (SEQID NO: 22).

“Example 3”

“Expression of the AAD Fusion Protein and its Purification”

“(3a). Expression of AAD Fusion Protein using Shake-Flask Method

“(3b). Expression of AAD Fusion Protein By Fermentation Method”

“(3c). Purification of AAD Fusion Protein”

After centrifugation, the soluble portion of the protein is collected. The nickel affinity chromatography is used to purify the fusion protein, which contains a His tag. TABLE 2 shows how the cultivation temperature affects the solubility AAD fusion proteins (amino acids sequence is shown in FIG. SEQ ID NO. 40, FIG. 3E) is obtained from the expression host.

The cell pellet should be resuspended in 25ml of 10mM sodiumphosphate buffer pH 7.4. The cells can be lysed using sonication and/or a high pressure homogenizer. After centrifugation, the soluble portion of the cells is separated. After centrifugation, the AAD fusion protein (with or without a His tag) can be purified using nickel affinity chromatography and/or Ion-Exchange columns.

The cell pellet can be used to isolate the insoluble fraction AAD fusion proteins. It is resuspended with 25 ml 20 mM TrisHCl, pH 7.4, 1% TRITON X100. The cells are lysed using sonication. Centrifugation is used to collect the insoluble portions (inclusion bodies). After the protein has been dissolved, it is resuspended in 10 ml 20 mM TrisHCl, pH 7.4, 6 m Guanidine HCl and vortexed to make it soluble. You can refold the protein by dropping it into 100 ml (20 mM) Sodium phosphate buffer pH 7.4. Centrifugation is used to remove insoluble substances. To achieve 70% saturation, salting the protein can be done by adding solid ammonium sulfurate powder to the supernatant. Centrifugation separates the insoluble part and it is then resuspended with 10 ml 20 mM sodiumphosphate buffer. The AAD fusion protein, which may contain a His tag (or not), is then purified using nickel affinity chromatography or ion-exchange columns.

TABLE 3 shows the yield and enzyme activity of AAD Fusion Protein from Shake-Flask Method and Fermentation Method.

“TABLE 3\nActivity\nAAD Yield (mg/L) (U/mg)\nShake-flask method ~10 ~9\nFermentation method ~42 ~19”

“Example 4”

“Enzyme Activity Assay & Enzyme Kinetics For AAD Fusion Protein”

“To determine the enzyme activity for wild-type ADI and AAD fusion protein in the present invention, the diacetyl monoxime (DAM)-thiosemicarbazide (TSC) assay for citrulline detection is used. The reaction is shown below.\nL-Arginine\nargininedeiminase(ADI)orAADfusion >L-Citrulline+Ammonia”

The Michaelis constant Km, which is an inverted measure of the substrate’s affinity to the enzyme, is the concentration at which reaction rate is at half-maximum. Km below 0.5 indicates a high affinity for the substrate. This means that the reaction rate will reach the maximum rate faster. The enzyme kinetics, or Km, of wild-type ADI (or AAD fusion protein) are determined under various concentrations of substrate arginine (2000??M, 1000??M, 500???M, 250??M), at pH 7.4. FIG. 3E shows the Km values for AAD fusion proteins. 3E (SEQID NO: 40), ADI protein is derived from Mycoplasma arginini. FIG. 3F (SEQID NO: 41), ADI protein is from Bacillus cereus), are 0.0041 mM, 0.132 mM, respectively. These results indicate that ABD fusion did not alter the binding affinity of different AAD fusion proteins for arginine.

“Example 5”

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