Cysteine ​​convertase modification and its application

Abstract

This invention provides an isolated or purified cysteine ​​convertase mutant and its application, specifically for the preparation of pharmaceutical compositions for treating cancer. Furthermore, this invention also provides a method for the in vitro degradation of cysteine, the method comprising the step of: in the presence of the cysteine ​​convertase mutant, causing cysteine ​​to be degraded in vitro to generate pyruvate.

Description

Technical Field

[0001] This invention belongs to the field of tumor therapeutic drugs, specifically involving the modification and application of cysteine ​​convertase. Background Technology

[0002] Malignant tumors seriously affect human health and are currently the second leading cause of death worldwide. In recent years, in addition to traditional surgical, radiotherapy, and chemotherapy methods, new treatments such as targeted therapy and immunotherapy have developed rapidly.

[0003] Currently, metabolic therapies based on the targeted degradation of specific amino acids highly needed by tumor cells are gradually becoming important cancer treatment methods. Among them, amino acid depletion therapy utilizes heterologous enzymes, recombinant enzymes, or engineered enzymes to target and inhibit the uptake of specific amino acids and promote their catabolism, which can effectively inhibit the growth of tumor cells. In addition, this therapy has far fewer side effects than radiotherapy and chemotherapy, and its impact on normal cells is negligible.

[0004] As one of the few sulfur-containing amino acids and a component of glutathione, cysteine ​​plays a crucial role in intracellular redox balance, in addition to its involvement in protein synthesis and post-translational modifications. When cystine-cysteine ​​levels are depleted within the cell, the production of reduced glutathione (GSH), an important intracellular antioxidant, decreases, severely disrupting the intracellular redox balance and leading to cell death. Tumor cells, due to genetic alterations and abnormal proliferation, experience higher levels of reactive oxygen species (ROS) oxidative stress than normal cells. Therefore, reduced glutathione (GSH), an intracellular antioxidant, is essential for cell survival and proliferation. Since cysteine ​​is a component of GSH, elevated GSH levels can deplete endogenous cysteine ​​sources. Although tumor cells can produce cysteine ​​from methionine via homocysteine, this is often insufficient to meet their needs. Therefore, tumor cells must absorb large amounts of exogenous cysteine ​​to survive.

[0005] Therefore, there is an urgent need in this field to develop a highly active cysteine ​​convertase to achieve efficient in vitro degradation of exogenous cysteine ​​in tumor cells and inhibit tumor growth. Summary of the Invention

[0006] The purpose of this invention is to provide a mutant of bacterial (cysteine) convertase and to use it in the preparation of a drug for treating cancer.

[0007] In a first aspect of the invention, an isolated or purified (cysteine) convertase mutant is provided, the mutant having the following site mutations: E43T, E321V, wherein the site is based on the amino acid sequence of the wild-type (cysteine) convertase shown in SEQ ID NO:1.

[0008] In another preferred embodiment, the amino acid sequence of the mutant is shown in SEQ ID NO:2.

[0009] In another preferred embodiment, the mutant has the following activity: in vitro catalytic degradation of cysteine.

[0010] In another preferred embodiment, the (cysteine)convertase mutant comprises its active fragment, variant form, or derived protein, wherein the active fragment, variant form, or derived protein has amino acid mutations of E43T and E321V, and has ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity with the (cysteine)convertase mutant, and has in vitro cysteine ​​degradation activity.

[0011] In another preferred embodiment, the mutant-derived protein comprises an amino acid sequence formed by amino acid mutations of E43T and E321V on the sequence shown in SEQ ID NO:2, which, after further having one or more amino acid residue deletions, insertions and / or substitutions, still has the activity of degrading cysteine ​​in vitro.

[0012] In another preferred embodiment, the plurality typically refers to 1-30, more preferably 1-10, even more preferably 1-6, further preferably 1-3, and most preferably 1.

[0013] In a second aspect of the invention, a separated polynucleotide is provided, said separated polynucleotide encoding the (cysteine ​​convertase mutant described in the first aspect of the invention.

[0014] In another preferred embodiment, the polynucleotide is selected from the group consisting of DNA, RNA, cDNA, or combinations thereof.

[0015] In a third aspect of the invention, a carrier is provided, the carrier containing the polynucleotide described in the second aspect of the invention.

[0016] In another preferred embodiment, the vector includes bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0017] In another preferred embodiment, the carrier is an expression carrier.

[0018] In another preferred embodiment, the vector is the pET28a plasmid.

[0019] In a fourth aspect of the invention, a host cell is provided, said host cell containing the vector described in the third aspect of the invention, or said host cell having the polynucleotide described in the second aspect of the invention integrated into its genome.

[0020] In another preferred embodiment, suitable host cells include Gram-positive bacteria, Gram-negative bacteria, actinomycetes, yeasts, and fungi. The Gram-positive bacteria include, but are not limited to, Bacillus subtilis; the Gram-negative bacteria include, but are not limited to, Escherichia coli; the actinomycetes include, but are not limited to, Streptomyces; the yeasts include, but are not limited to, Saccharomyces cerevisiae; and the fungi include, but are not limited to, Aspergillus. Their cells are all commonly used host cells for recombinant vectors.

[0021] In another preferred embodiment, the host cell also contains a vector for expressing the fusion protein or a protein mutant integrated into the chromosome.

[0022] In another preferred embodiment, the host cell expresses a protein mutant.

[0023] In a fifth aspect of the invention, a method for preparing the (cysteine ​​convertase mutant described in the first aspect of the invention is provided, comprising the steps of:

[0024] (i) Under suitable expression conditions, host cells as described in the fourth aspect of the present invention are cultured to express the (cysteine ​​convertase mutant as described in the first aspect of the present invention;

[0025] (ii) Isolate the expression product to obtain the (cysteine) convertase mutant.

[0026] In a sixth aspect of the invention, a method for in vitro degradation of (cysteine) is provided, comprising the steps of:

[0027] In the presence of the (cysteine) convertase mutant described in the first aspect of this invention, cysteine ​​is degraded in vitro to generate pyruvate:

[0028]

[0029] In another preferred embodiment, the method for in vitro degradation of (cysteine) is a method for in vitro degradation of (cysteine) for non-diagnostic and therapeutic purposes.

[0030] In another preferred embodiment, the (cysteine) includes (but is not limited to): homocysteine, cystine, and cysteine.

[0031] In a seventh aspect of the invention, a reaction system for the in vitro degradation of cysteine ​​is provided, the reaction system comprising:

[0032] (i) the (cysteine) convertase mutant described in the first aspect of the present invention; and

[0033] (ii) (cysteine) substrate.

[0034] In another preferred embodiment, the (cysteine) substrate includes: cystine, cysteine, homocysteine, or a combination thereof.

[0035] In an eighth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0036] (i) a pharmaceutically acceptable carrier; and

[0037] (ii) A (cysteine) convertase mutant as described in the first aspect of the invention or a polynucleotide as described in the second aspect of the invention.

[0038] In a ninth aspect of the invention, the use of the (cysteine ​​convertase mutant described in the first aspect of the invention, the polynucleotide described in the second aspect of the invention, or the pharmaceutical composition described in the eighth aspect of the invention is provided for use in the preparation of a pharmaceutical composition for treating cancer.

[0039] In another preferred embodiment, the cancer may be a solid cancer.

[0040] In another preferred embodiment, the solid cancer includes lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma, and other solid tumors.

[0041] In another preferred embodiment, the cancer may be a liquid cancer.

[0042] In another preferred embodiment, the liquid cancer includes non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors.

[0043] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0044] Figure 1 The components and functions of the cystine / glutamate transporter system are shown.

[0045] Figure 2 The BacCyse expression recombinant plasmid was displayed.

[0046] Figure 2 A shows the map of the recombinant plasmid Cyst(e)inase; Figure 2 B shows the enzyme digestion verification results.

[0047] Figure 3 The expression and purification of the BacCyse protein are shown.

[0048] Figure 3 A shows the electrophoresis diagram of BacCyse protein after induction with 1 mM IPTG at 37°C for 16 hours; Figure 3 B shows the purity and concentration of the purified BacCyse protein; Figure 3 C shows the protein purity of BacCyse as determined by Image J analysis; Figure 3 D shows the BacCyse protein concentration obtained from Image J analysis.

[0049] Figure 4 The results showed that the BacCyse protein can degrade cysteine.

[0050] Figure 4 A shows the results of incubating at 37°C for 2 hours with 2.5 mM cysteine ​​as a substrate, adding 1 μg / mL BacCyse protein to the experimental group and an equal volume of PBS to the NT group, and then measuring the OD. 600 . Figure 4 B shows the OD values ​​measured at different time points (30 min, 2 h, 8 h, 32 h, and 56 h) using 2 mM cysteine ​​as a substrate, with 10 μg / mL BacCyse protein added, and the control group with an equal volume of PBS. 600 .

[0051] Figure 5 This shows that Cyst(e)inase protein has a stronger cysteine-degrading ability compared to McB protein.

[0052] Figure 5 A shows the remaining cysteine ​​concentrations in the control group, Cyst(e)inase group, MccB group, Cyst(e)inase + heat inactivation group, and MccB + heat inactivation group after degradation of 1 mM cysteine ​​standard at 37°C for 2 h. Figure 5 B shows the relative content of remaining cysteine ​​in the control group, Cyst(e)inase group, MccB group, Cyst(e)inase + heat inactivation group, and MccB + heat inactivation group after degradation of 1 mM cysteine ​​standard at 37°C for 2 h.

[0053] Figure 6 The results showed that BacCyse protein inhibits the growth of HCT116 cells.

[0054] Figure 6 A shows that the initial number of HCT116 cells per well was 7*10. 4After culturing at 37°C and 5% CO2 for 72 hours, the cells were photographed and their condition observed. The corresponding amount of PBS was added to the NT group. Figure 6 B shows the proportion of cells at a concentration of 1 μg / mL (cysteine) protein compared to the control group (NT) after 72 hours. All studies were performed in triplicate, with three replicates per group. ***, p < 0.001.

[0055] Figure 7 The results showed that the BacCyse protein can inhibit the growth of LN229 cells.

[0056] Figure 7 A shows that the initial number of LN229 cells per well was 7.5*10. 4 Cells were cultured at 37℃ and 5% CO2 for 24, 48, and 72 hours, and their condition was observed by photographing. The NT group was treated with the corresponding amount of PBS, while the drug group was treated with 10ug / ml of BacCyse protein. Figure 7 B shows the proportion of cells in the BacCyse protein treatment group compared to the control group (NT) after 24 hours, 48 ​​hours, and 72 hours. Figure 7 C shows the changes in cell counts in the BacCyse protein treatment group and the control group (NT) after 24, 48, and 72 hours. FM - control group, complete culture medium. FM - (cysteine) enzyme, treated with 10 μg / ml BacCyse protein in complete culture medium. All studies were performed in triplicate, with three replicates per group per replicate.

[0057] Figure 8 The results showed that BacCyse protein inhibits the growth of MC38 cells.

[0058] Figure 8 A shows that the initial number of MC38 cells per well was 8*10. 4 Cells were cultured at 37℃ and 5% CO2 for 24, 48, and 72 hours, and their condition was observed by photographing. The NT group was supplemented with the corresponding amount of PBS, and the (cysteine) group was supplemented with 10ug / ml BacCyse protein. Figure 8 B shows the proportion of cells in the BacCyse protein treatment group compared to the control group (NT) after 24 hours, 48 ​​hours, and 72 hours. Figure 8 C shows the changes in cell counts in the BacCyse protein treatment group and the control group (NT) after 24, 48, and 72 hours. FM - control group, complete culture medium. FM - (cysteine) enzyme, complete culture medium with BacCyse protein added. All studies were performed in triplicate, with three replicates per group per replicate.

[0059] Figure 9 This diagram illustrates in vivo treatment in a mouse tumor-bearing model. 6-8 week old C57 / 6j mice were injected with MC38-1*10 in the right thigh. 6 The NT group received intraperitoneal injections of the corresponding amount of PBS on days 7, 9, 11, and 14 after subcutaneous tumor formation. The experimental group received intraperitoneal injections of BacCyse protein on days 7, 9, 11, and 14 after subcutaneous tumor formation, while the IR group received radiotherapy on day 9 after subcutaneous tumor formation.

[0060] Figure 10 The results showed that BacCyse protein inhibited tumor growth in mice. NT (n=5), IR (10Gray; n=6), (cysteine)ase (100 mg / kg BacCyse protein; n=5). *, p<0.05. Detailed Implementation

[0061] Through extensive and in-depth research, including numerous screenings and studies of mutant proteins, the inventors unexpectedly obtained a mutant of a bacterial cysteine ​​convertase (BacCyse, Bacterial L-cyst(e)inase). They discovered that this cysteine ​​convertase mutant can efficiently degrade extracellular cysteine, thereby reducing the extracellular cysteine ​​concentration in tumor cells and effectively inhibiting the growth and proliferation of various cancer cells. This invention was completed based on this finding.

[0062] Specifically, this invention clones the natural mccB gene from Bacillus subtilis, a common species in food and the environment. The protein sequence is then modified by mutating glutamic acid at position 43 to threonine (E43T) and glutamic acid at position 321 to valine (E321V), ultimately yielding a novel (cysteine) hydrolase named BacCyse.

[0063] The mutated cysteine ​​convertase exhibits approximately a 5-fold increased activity in degrading cysteine ​​and can also directly degrade it to pyruvate. The cysteine ​​convertase mutant of this invention can effectively inhibit tumor growth in mice, with initial effects comparable to irradiation.

[0064] As used herein, “(cysteine)transferase,” “wild-type (cysteine)transferase,” “cysteine ​​transferase,” “wild-type cysteine ​​transferase,” or “wild-type McB protein” are used interchangeably and all refer to (cysteine)transferase. It should be understood that the (cysteine)transferase referred to here refers to an enzyme that converts cysteine ​​or cystine.

[0065] In this invention, the terms "(cysteine)convertase mutant", "mutant (cysteine)convertase", "cysteine ​​convertase mutant", "mutant", "mutant protein", "active polypeptide of the present invention", "(cysteine)ase", "Cyst(e)inase" or "BacCyse protein" are used interchangeably and refer to the (cysteine)convertase mutant described in the first aspect of this invention.

[0066] Existing engineered enzymes for reducing cysteine ​​are mainly derived from eukaryotes, but eukaryotic protein expression systems have drawbacks such as numerous procedures and high costs. In addition, the codons of eukaryotic proteins are often rare in prokaryotes, resulting in lower yields and easy formation of inclusion bodies in the low-cost E. coli expression system.

[0067] This invention relates to a prokaryotic-derived cysteine ​​convertase with an enzyme sequence of only 379 amino acids, making it easy to express and purify on a large scale in engineered bacteria, and suitable for industrial fermentation and production. Furthermore, the mutant obtained by this invention exhibits superior cysteine ​​degradation activity and stronger inhibitory ability against cancer cell growth in vitro.

[0068] The microbial gene yrhB / mccB encodes a wild-type cysteine ​​convertase, named mccB (methionine-to-cysteine ​​conversion) because it promotes the conversion of methionine to cysteine. This enzyme is widely found in prokaryotic bacteria and shares 50% similarity with human cystathionine-γ-lyase, but is shorter (only 379 amino acids), making it easier to express, ferment, and purify on a large scale in engineered bacteria.

[0069] Wild-type bacterial MccB protein exhibits cystathionine-lyase and homocysteine-γ-lyase activities in vitro. MccB shares 50% similarity with cystathionine β-hydrolase (MetC) and 46% similarity with cystathionine-γ-hydrolases from rat and Saccharomyces cerevisiae. The purified MccB enzyme activity follows Michaelis-Menten kinetics, with a Km value of approximately 3 mM, and produces approximately 2 μmol of free thiol groups (V2) per milligram of protein per minute. max Cysteine ​​γ-hydrolase degrades cysteine ​​to produce compounds containing free thiols, namely homocysteine ​​or cysteine.

[0070] Tumor cells can accelerate the uptake of extracellular cysteine ​​by upregulating the cysteine / glutamate reverse transport protein (xCT transporter). For example... Figure 1 As shown, cystine taken up from outside the cell reaches the cell and is partially converted into cysteine, thereby increasing GSH synthesis.

[0071] Tumor cells often rely on the SLC7A11 / SLC3A2 transport system to absorb cystine, and then convert it into cysteine ​​through NADPH-dependent reduction reactions for the synthesis of biomolecules such as glutathione and proteins.

[0072] In a specific embodiment, the wild-type (cysteine) convertase and the gene encoding it are derived from prokaryotes; preferably, from bacteria; more preferably, from Bacillus subtilis.

[0073] In a specific embodiment, the amino acid sequence of the wild-type (cysteine) convertase is shown in SEQ ID NO:1:

[0074] MKKKTLMIHGGITGDEKTGAVSVPIYQVSTYKQPKAGQHTGYEYSRTANPTRTALEALVTELESGEAGYAFSSGMAAITAVMMLFNSGDHVVLTDDVYGGTYRVMTKVLNRLGIESTFVDTSSREEVEKAIRPNTKAIYIETPTNPLLKITDLTLMADIAKKAGVLLIVDNTFNTPYFQQPLTLGADIVL HSATKYLGGHSDVVGGLVVTASKELGEELHFVQNSTGGVLGPQDSWLLMRGIKTLGLRMEAIDQNARKIASFLENHPAVQTLYYPGSSNHPGHELAKTQGAGFGGMISFDIGSEERVDAFLGNLKLFTIAESLGAVESLISVPARMTHASIPRERRLELGITDGLIRISVGIEDAEDLLEDIGQALENI*.

[0075] In a specific embodiment, the (cysteine) convertase includes its active fragment, variant form, or derived protein, wherein the active fragment, variant form, or derived protein has amino acids E43 and E321, and has ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% sequence identity with the wild-type (cysteine) convertase, and has in vitro cysteine ​​degradation activity.

[0076] In a specific embodiment, the derived protein comprises an amino acid sequence having amino acids E43 and E321 on the sequence shown in SEQ ID NO:1, and further having one or more (e.g., typically 1-30, preferably 1-10, more preferably 1-6, even more preferably 1-3, most preferably 1) amino acid residues deleted, inserted and / or substituted, and still having in vitro cysteine ​​degradation activity.

[0077] In a specific embodiment, the mutant of the (cysteine) convertase is a (cysteine) convertase mutant with the following mutations: E43T, E321V, wherein the site is based on the amino acid sequence of the wild-type (cysteine) convertase shown in SEQ ID NO:1.

[0078] In a specific implementation, the mutant (cysteine) convertase can catalyze the efficient in vitro degradation of exogenous cysteine ​​in tumor cells, thereby inhibiting tumor cell growth, and its activity is significantly improved compared to the wild type.

[0079] In a specific embodiment, the mutant of the (cysteine) convertase has the following mutated (cysteine) convertase mutants: E43T, E321V, wherein the site is based on an amino acid sequence that exhibits at least 90% homology with the wild-type (cysteine) convertase shown in SEQ ID NO:1.

[0080] In a specific implementation, the mutant can degrade exogenous cysteine ​​in tumor cells in vitro, thereby inhibiting tumor cell growth and exhibiting significantly enhanced activity compared to the wild type.

[0081] In a specific embodiment, the amino acid sequence of the mutant (cysteine) convertase of the present invention is shown in SEQ ID NO:2:

[0082] MKKKTLMIHGGITGDEKTGAVSVPIYQVSTYKQPKAGQHTGY TYSRTANPTRTALEALVTELESGEAGYAFSSGMAAITAVMMLFNSGDHVVLTDDVYGGTYRVMTKVLNRLGIESTFVDTSSREEVEKAIRPNTKAIYIETPTNPLLKITDLTLMADIAKKAGVLLIVDNTFNTPYFQQP LTLGADIVLHSATKYLGGHSDVVGGLVVTASKELGEELHFVQNSTGGVLGPQDSWLLMRGIKTLGLRMEAIDQNARKIASFLENHPAVQTLYYPGSSNHPGHELAKTQGAGFGGMISFDIGSEERVDAFLGNLKLFTIA V SLGAVESLISVPARMTHASIPRERRLELGITDGLIRISVGIEDAEDLLEDIGQALENI*.

[0083] In specific embodiments, the mutant protein of the present invention further includes fragments, derivatives, and analogs of the mutant shown in SEQ ID NO:2, wherein the fragments, derivatives, and analogs substantially retain the activity of the (cysteine)convertase mutant of the present invention, and may be (i) a polypeptide with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, or (ii) a polypeptide having substituent groups in one or more amino acid residues, or (iii) a polypeptide formed by fusing the polypeptide of the present invention with another compound (such as a compound that prolongs the half-life of the polypeptide, for example polyethylene glycol), or (iv) a polypeptide formed by fusing an additional amino acid sequence to this polypeptide sequence (a fusion protein formed by fusing with a leader sequence, secretion sequence, or tag sequence such as His). These fragments, derivatives, and analogs are well known to those skilled in the art.

[0084] In specific embodiments, the mutant protein of the present invention also includes variant forms and derived polypeptides having the same function as the polypeptide shown in SEQ ID NO:2. These variant forms include, but are not limited to, the deletion, insertion, and / or substitution of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus.

[0085] In a specific embodiment, the present invention also provides analogues of the mutant.

[0086] In specific implementations, these mutants include natural or induced genetic variants. Induced variants can be obtained through various techniques, such as random mutagenesis through radiation or exposure to mutagens, or through site-directed mutagenesis or other known molecular biology techniques.

[0087] In specific embodiments, analogs also include analogs having residues different from naturally occurring L-amino acids (such as D-amino acids), and analogs having non-naturally occurring or synthetic amino acids (such as β- or γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above.

[0088] In specific embodiments, the amino or carboxyl terminus of the protein of the present invention may further contain one or more polypeptide fragments as protein tags. Any suitable tag can be used in the present invention. For example, representative tags include, but are not limited to, FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags can be used for protein purification.

[0089] In a specific embodiment, to enable the translated protein to be expressed secretively (e.g., secreted extracellularly), a signal peptide sequence, such as pelB signal peptide, can be added to the amino terminus of the (cysteine ​​convertase mutant). The signal peptide can be cleaved during the secretion of the polypeptide from the cell.

[0090] This invention relates to polynucleotides encoding mutants of this invention.

[0091] In specific embodiments, the polynucleotides of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0092] In a specific embodiment, the polynucleotide encoding the mature polypeptide of SEQ ID NO:2 includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0093] In a specific implementation, a "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or it may include a polynucleotide with additional coding and / or non-coding sequences.

[0094] In specific embodiments, the present invention also relates to variants of the aforementioned polynucleotides that encode polypeptides or fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as those of the present invention. These polynucleotide variants can be naturally occurring isoforms or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes.

[0095] In specific embodiments, the present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that are hybridizable with the polynucleotides described herein under stringent conditions (or strict conditions). In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO:2.

[0096] In specific embodiments, the present invention also relates to nucleic acid fragments that hybridize with the above-described sequences. As used herein, a “nucleic acid fragment” is at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more. The nucleic acid fragment can be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding (cysteine) mutants.

[0097] In specific embodiments, the polypeptides and polynucleotides of the present invention are preferably provided in isolated form, and more preferably purified to homogenization. The full-length sequence or fragment of the (cysteine)ase mutant of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. Once the relevant sequence is obtained, it can be obtained in large quantities by recombination. This typically involves cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods.

[0098] In specific implementations, the relevant sequences can also be synthesized artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then connecting them.

[0099] In specific embodiments, the DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis. The method of amplifying DNA / RNA using PCR technology is preferred for obtaining the gene of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA end amplification) is preferred. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0100] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells genetically engineered using the vectors of the present invention or (cysteine) mutant coding sequences, and methods for generating the mutants of the present invention via recombination technology.

[0101] In specific embodiments, the polynucleotide sequence of the present invention can be used to express or produce recombinant (cysteine)ase mutants using conventional recombinant DNA technology. Generally, the steps include: transforming or transducing suitable host cells with the polynucleotide (or variant) encoding the (cysteine)ase mutant of the present invention, or with a recombinant expression vector containing the polynucleotide; culturing the host cells in a suitable culture medium; and isolating and purifying the protein from the culture medium or cells.

[0102] In a specific implementation, the (cysteine)ase mutant polynucleotide sequence can be inserted into the recombinant expression vector. The "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0103] In specific embodiments, those skilled in the art can construct expression vectors containing a (cysteine)ase mutant encoding DNA sequence and suitable transcription / translation control signals using well-known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the *E. coli* lac or *trp* promoter; the *λ* phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTRs, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0104] In a specific embodiment, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0105] In a specific implementation, a vector containing the aforementioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express a protein.

[0106] In specific implementations, the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; Salmonella typhimurium bacterial cells; fungal cells such as yeast; plant cells; Drosophila S2 or Sf9 insect cells; and animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells.

[0107] In specific implementations, suitable host cells include Gram-positive bacteria such as Bacillus subtilis, Gram-negative bacteria such as Escherichia coli, actinomycetes such as Streptomyces, yeasts such as Saccharomyces cerevisiae, and fungi such as Aspergillus. Their cells are all commonly used host cells for recombinant vectors.

[0108] In a specific embodiment, when the polynucleotide of the present invention is expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0109] In specific implementations, those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0110] In specific implementations, transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0111] In a specific embodiment, the obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0112] In specific embodiments, the recombinant polypeptides in the above methods can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0113] The present invention also provides a reaction system for the in vitro degradation of (cysteine). The reaction system comprises (i) the wild-type (cysteine) convertase or a cysteine ​​convertase mutant described in this invention and (ii) a cysteine ​​substrate.

[0114] As used herein, the cysteine ​​substrate includes those selected from homocysteine, cystine, cysteine, or combinations thereof.

[0115] application

[0116] The present invention also provides a pharmaceutical composition comprising (i) a pharmaceutically acceptable carrier and (ii) a (cysteine)ase mutant as described in the first aspect of the present invention or a polynucleotide as described in the second aspect of the present invention.

[0117] As used herein, the term "carrier" means any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation, or other material well known in the art for use in pharmaceutical formulations. It should be understood that the properties of the carrier, excipient, or diluent will depend on the route of administration in the specific application. As used herein, the term "pharmaceuticalally acceptable carrier" means a non-toxic material that does not interfere with the effectiveness or bioactivity of the compositions according to the invention.

[0118] In specific embodiments, the pharmaceutical composition may be in any suitable form, depending on the patient's preferred method of administration. It may be provided in unit dosage forms, typically in a sealed container, and may be offered as part of a kit. Such kits typically (but are not required to) include instructions for use. They may contain multiple of the aforementioned unit dosage forms.

[0119] In specific embodiments, the pharmaceutical composition is suitable for any suitable route of administration, such as injection (including subcutaneous, intradermal, intramuscular, intraperitoneal, microneedle, or intravenous injection), inhalation or oral administration, or via the nose or anus. The composition can be prepared by any method known in the pharmaceutical field, for example, under aseptic conditions by mixing the active ingredient with a carrier or excipient.

[0120] The present invention also provides the use of the (cysteine)ase mutant of the present invention, the polynucleotide of the present invention, and the pharmaceutical composition of the present invention for the preparation of a pharmaceutical composition for treating cancer.

[0121] The cancer can be any liquid or solid cancer, including, but not limited to, lung cancer, stomach cancer, esophageal cancer, bile duct cancer, bile duct epithelial cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder transitional cell carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and liquid tumors.

[0122] As used herein, "isolated" means that a biological composition (e.g., nucleic acid, peptide, or protein) has been substantially isolated, produced, or purified from other biological components naturally present in the organism, including other chromosomal and extrachromosomal DNA and RNA, as well as proteins. Therefore, "isolated" nucleic acids, peptides, and proteins include nucleic acids and proteins purified using standard purification methods. "Isolated" nucleic acids, peptides, and proteins can be part of a complex, and if the complex is not part of the native environment of the nucleic acid, peptide, or protein, the nucleic acid, peptide, or protein is still isolated. The term also includes nucleic acids, peptides, and proteins prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acids.

[0123] As used herein, the terms “peptide,” “polypeptide,” or “protein” can refer to a molecule composed of amino acids that can be considered a protein by those skilled in the art. This invention uses conventional encoding methods for amino acid residues, i.e., one-letter or three-letter encodings. The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymers can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acid groups. The term also includes amino acid polymers that have been naturally modified or modified through intervention; said modification methods include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification, such as binding to a label. The definition of the term also covers, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids), and other modifications known in the art.

[0124] Modifications (which typically do not alter the primary structure) include: chemically derived forms of peptides, either in vivo or in vitro, such as acetylation or carboxylation. Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine), and peptide forms modified to improve their resistance to proteolytic hydrolysis or optimize their solubility.

[0125] The peptide sequences described in this invention are written according to common convention, i.e., the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although amino acid isomers are known, the L-form of the amino acid is represented unless otherwise explicitly indicated.

[0126] In specific implementations, for example, in the relevant art, substitution with amino acids of similar or identical properties generally does not alter the protein's function. Similarly, adding one or more amino acids to the C-terminus and / or N-terminus generally does not change the protein's function.

[0127] The active polypeptides of the present invention can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. The polypeptides of the present invention can be naturally purified products, chemically synthesized products, or produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, plants) using recombinant technology. Depending on the host used in the recombinant production protocol, the polypeptides of the present invention can be dehydrated or non-dehydrated. The polypeptides of the present invention may or may not include an initial methionine residue.

[0128] Advantages of the present invention

[0129] 1. The present invention obtained the (cysteine) convertase mutant of the present invention through extensive screening and targeted mutation. It was unexpectedly discovered that the obtained (cysteine) convertase mutant can efficiently degrade (cysteine) in vitro. The enzyme activity of the mutant is about five times that of the wild-type (cysteine) convertase.

[0130] 2. The (cysteine) convertase mutant described in this invention can significantly inhibit the proliferation of tumor cells.

[0131] 3. The (cysteine) convertase mutant described in this invention can significantly inhibit tumor growth.

[0132] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. Specific Implementation

[0134] Experimental animals: C57BL / 6J genetic background mice.

[0135] Vector plasmid: pET28a.

[0136] The cell lines included: human colorectal adenocarcinoma cells (HCT116); human glioblastoma cells (LN229); and mouse colon cancer cells (MC38), all purchased from ATCC.

[0137] Buffer formulation:

[0138]

[0139] Experimental methods:

[0140] a) Culture of mouse MC38 colon cancer cell line

[0141] MC38 mouse colon cancer cells are mainly used for in vitro cell proliferation experiments and the construction of mouse tumor-bearing models. MC38 culture medium is DMEM containing 10% FBS and 1% penicillin / streptomycin. Culture conditions are 37°C and saturated humidity of 5% CO2.

[0142] b) Culture of HCT116 human colon cancer cell line

[0143] Human colorectal cancer cells—HCT116—were primarily used for in vitro cell proliferation experiments. HCT116 cells were cultured in McCoy's 5A medium containing 10% FBS and 1% penicillin and streptomycin. Culture conditions were 37°C and saturated humidity with 5% CO2.

[0144] c) Culture of LN229 neuroblastoma cell line

[0145] Human neuroblastoma cells—LN229—were primarily used for in vitro cell proliferation experiments. LN229 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin and streptomycin. Culture conditions were 37°C and saturated humidity with 5% CO2.

[0146] d) Mouse tumor-bearing model

[0147] Wild-type mice with a C57BL / 6J genetic background, aged 6-8 weeks, were anesthetized by intraperitoneal injection of aphthylamine. The right thigh of the mice was then shaved to expose the skin. A syringe with a 0.33*13mm needle was used to inject 1*10... 6 MC38 mouse colon cancer cells were subcutaneously inoculated. To avoid leakage of the injection solution, the needle was carefully removed 3 seconds after injection.

[0148] e) Mouse treatment model

[0149] After successful subcutaneous injection of MC38 into mice to establish the tumor model, wait until the subcutaneous tumor size of the mice reaches 100-200 mm. 3 During this period, tumor-bearing mice were randomly divided into groups and administered different doses of the protein drug via intraperitoneal injection using a 1 mL syringe. Tumor measurements were then taken every 2-3 days. Depending on requirements, some tumor-bearing mice were irradiated before drug administration to evaluate the efficacy of the combined treatment.

[0150] f) Cyst(e)inase protein purification method

[0151] (f1) Transform the BacCyse protein recombinant plasmid into BL21(DE3) competent E. coli cells, incubate on ice for 30 minutes, place in a 42°C water bath for 1 minute, and then quickly freeze on ice for 2-5 minutes. Add 1 mL of LB medium (antibiotic-free) to the competent cells and incubate at 37°C, 220 rpm for 1 hour. Plate the cells and incubate at 37°C for 16 hours.

[0152] (f2) Pick a single colony and place each one into a 1.5 mL EP tube. Add 600 uL LB and incubate at 37°C and 220 rpm for 4-8 hours to allow it to reach the logarithmic growth phase.

[0153] (f3) Add the bacterial culture that has reached the logarithmic growth phase to a 15 mL shake tube, add 8 mL of LB medium (containing kanamycin resistance), incubate at 37°C and 220 rpm for 16 hours, and take a portion of the bacterial culture for glycerol preservation.

[0154] (f4) Add the bacterial culture from the 15 mL shaker tube to 1 L LB medium (containing kanamycin resistance), incubate at 37 °C and 220 rpm for 2-3 hours. Induce OD600 at 0.6.

[0155] (f5) Take 1 mL and store it in a 1.5 mL EP tube as a pre-induction control. Add the remaining bacterial culture to a final concentration of 1 mM IPTG for induction at 25°C and 220 rpm for 16 hours. After induction, take 1 mL and store it in a 1.5 mL EP tube as a post-induction control.

[0156] (f6) Centrifuge the induced bacterial culture at 6000 rpm for 10 minutes, add 40 mL of lysis buffer and protease inhibitor, and sonicate for 40% duty, 5 s on, 3 s off, for 30 minutes. After disruption, centrifuge at 17000 rpm for 30 minutes. Collect the supernatant and filter it through a 0.45 μm filter membrane. Use the filtered liquid for the next experiment.

[0157] (f7) For Ni-NTA columns, mix the supernatant with the Ni column and mix at 4°C for 1 hour. Place the column vertically and let it stand for 10 minutes. Collect the eluent. First, resuspend the Ni column with 1 column volume of elution buffer for contaminating proteins. Repeat this process two or three times. Then, wash the column with a total of 10 column volumes of elution buffer for contaminating proteins.

[0158] (f8) First, resuspend the Ni column with 1 column volume of the target protein elution buffer, repeat two or three times, and then collect the target protein with 5 column volumes of the target protein elution buffer.

[0159] (f9) The collected target protein eluent was placed in an activated dialysis bag and placed in pre-cooled PBS for replacement buffering at 4°C for 24 hours. Finally, the target protein was detected by SDS-PAGE and its concentration was determined.

[0160] g) Cyst(e)inase protein endotoxin removal

[0161] (g1) All items are removed without a pyrogen source. Containers are soaked in 0.5M NaOH for 30 minutes and then washed with ultrapure water without a pyrogen source.

[0162] (g2) Add 0.5% Triton X-114 to the lysis buffer. Add it during ultrasonic disruption. When rinsing the nickel column, use the lysis buffer containing Triton X-114 and then rinse with the lysis buffer without Triton X-114.

[0163] (g3) The prepared solution should be stored at 4°C.

[0164] (g4) Use equipment such as pyrogen-free EP tubes, pyrogen-free ultrapure water, and pyrogen-free 0.22um filter membranes.

[0165] h) Statistical Analysis

[0166] In this invention, all quantitative data are expressed as mean plus standard error (SEM). Significant differences were determined using a T-test. A p-value less than 0.05 was considered statistically significant. Data were obtained from at least three independent experiments, with at least three replicates in each experiment.

[0167] Example 1: Construction of BacCyse protein expression plasmid

[0168] A BacCyse expression recombinant plasmid was constructed based on the pET-28a plasmid vector. The constructed plasmid is as follows: Figure 2 As shown in Figure A, the recombinant plasmid was digested with a single enzyme (BamHI) and then with two enzymes (BamHI and XhoI), followed by agarose gel electrophoresis analysis.

[0169] Agarose gel electrophoresis results are as follows Figure 2 As shown in Figure B, the figure presents a single band of approximately 6500 bp (single enzyme digestion) and two double bands of approximately 5500 bp and 1100 bp (double enzyme digestion), confirming the successful construction of the BacCyse protein expression plasmid.

[0170] Example 2: BacCyse protein expression and purification

[0171] After transforming the recombinant plasmid into E. coli, BacCyse protein was expressed and purified. The specific steps were as follows: after induction with 1 mM IPTG at 37°C for 16 hours, the bacteria were collected by centrifugation, and the supernatant was collected after sonication; SDS-PAGE electrophoresis was followed by Coomassie brilliant blue staining. Figure 3 A high level of expression of the target protein was observed in sample A. Subsequently, the His-tagged BacCyse protein was purified using a Ni column; the protein solubility buffer was replaced with phosphate buffer via a dialysis bag to finally obtain the BacCyse protein. A small amount of protein was subjected to SDS-PAGE and stained with Coomassie Brilliant Blue, as shown in the results. Figure 3 As shown in B.

[0172] Depend on Figure 3 C and Figure 3 The results of comparison between D and the protein standard BSA show that the purity of BacCyse protein is approximately 93% and the concentration is approximately 2.5 mg / mL.

[0173] Example 3: Compared to wild-type protein, BacCyse protein exhibits higher enzymatic activity in degrading cysteine.

[0174] Cysteine ​​contains a sulfhydryl group, which can reduce phosphate to produce tungsten blue. The product is present in the OD. 600 It has a maximum absorption peak, therefore, by detecting OD 600 Absorbance reflects the reduction of cysteine ​​to phosphate, indirectly detecting the degradation of cysteine ​​by BacCyse protein.

[0175] The results are as follows Figure 4 As shown in Figure A, under the same cysteine ​​substrate concentration, 1 μg / mL of BacCyse protein can degrade 20% of cysteine, indicating that the BacCyse protein of the present invention has a strong affinity for cysteine ​​and can significantly reduce the concentration of cysteine ​​substrate.

[0176] Figure 4 The results of B showed that 10 μg / mL BacCyse protein could significantly reduce the content of cysteine ​​substrate after 30 minutes, decompose about half of the substrate in about 4 hours, and the decomposition reaction tended to stabilize after 20 hours, indicating the high efficiency of BacCyse protein in in vitro degradation of cysteine.

[0177] To investigate the cysteine-degrading activities of the unmutated MccB protein and the mutated BacCyse protein, 10 μg / mL BacCyse and MccB proteins were reacted with 1 mM cysteine ​​standard at 37°C for 2 h. The absorbance was analyzed using a cysteine ​​content detection kit. The control group was treated at 95°C for 5 minutes.

[0178] Figure 5 A and Figure 5 The results of B showed that both MccB and BacCyse proteins have the ability to degrade cysteine. Under the same substrate concentration, the same enzyme content, and the same reaction time, Cyst(e)inase protein has a stronger cysteine ​​degradation ability than MccB protein, and its enzyme activity efficiency is about 5 times that of MccB protein.

[0179] After heat inactivation treatment, the ability of McB protein and BacCyse protein to degrade cysteine ​​was lost.

[0180] Example 4: BacCyse protein significantly inhibits tumor cell growth

[0181] After treating HCT116 cells with different concentrations of BacCyse protein for 72 hours, the cell growth was as follows: Figure 6 As shown in Figure A.

[0182] Depend on Figure 6 As shown in Figure B, 1 μg / mL of BacCyse protein significantly inhibited the growth of the human colon cancer cell line HCT116, and the degree of inhibition was positively correlated with the concentration of BacCyse protein used. This indicates that BacCyse protein can significantly and efficiently inhibit the growth of the human colon cancer cell line HCT116.

[0183] After adding 10 μg / mL of BacCyse protein to human glioblastoma LN229 cells for 24, 48, and 72 hours, changes in cell number and cell growth were detected. Figure 7 As shown in Figure A.

[0184] Depend on Figure 7 B and Figure 7 As shown in Figure C, after 48 and 72 hours of treatment with 10 μg / mL BacCyse protein, the number of human glioblastoma LN229 cells was significantly reduced compared to the PBS-treated control group. Two days of BacCyse protein treatment significantly inhibited the growth of LN229 cells. After 48 hours of treatment with 10 μg / mL BacCyse protein, the cell number of human glioblastoma LN229 cells decreased by more than 50%. This indicates that BacCyse protein can significantly and efficiently inhibit the growth of human glioblastoma LN229 cells.

[0185] To determine whether BacCyse protein could exert an effect on the mouse colon cancer MC38 cell line, cell growth was assessed after 24, 48, and 72 hours of BacCyse protein treatment. Figure 8 As shown in Figure A.

[0186] Figure 8 B and Figure 8 C indicates that when mouse colon cancer MC38 cell lines were treated with 10 μg / mL BacCyse protein for 24 hours, the cell number was reduced by approximately 40% compared to the control group; after 48 hours of treatment, the cell number was reduced by approximately 75% compared to the control group; and after 72 hours of treatment, the cell number was reduced by approximately 90%, indicating that BacCyse protein can significantly and efficiently inhibit the growth of mouse colon cancer MC38 cell lines.

[0187] The data in this embodiment show that the BacCyse protein of the present invention can significantly and efficiently inhibit the growth of various cancer cells.

[0188] Example 5: BacCyse protein significantly inhibits MC38 tumor growth in tumor-bearing mice.

[0189] This study investigated whether the BacCyse protein could inhibit tumor growth in MC38 tumor-bearing mice. First, a subcutaneous tumor-bearing model was established by inoculating the right leg of 6-8 week old mice with the MC38 colon cancer cell line. (The text repeats itself here.) Figure 9 As shown, (cysteine) protein was injected intraperitoneally starting from the seventh day of tumor bearing in mice, with injections every two to three days, for a total of four administrations. To better demonstrate the therapeutic effect of (cysteine) protein in the mouse tumor model, radiotherapy, a currently mature clinical tumor treatment method, was selected as a positive control experiment, and radiotherapy was administered to the mice on the ninth day of tumor bearing.

[0190] The results are as follows Figure 10 As shown, continuous measurement of tumor size demonstrated that BacCyse protein alone can significantly inhibit tumor growth in vivo; within two days after the last injection of BacCyse protein, the inhibitory effect on tumors was comparable to that of radiotherapy, and thereafter the effect was slightly weaker than that of radiotherapy, but there was still a significant difference compared with the control group.

[0191] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An isolated or purified cysteine ​​convertase mutant, characterized in that, The mutants described are cysteine ​​convertase mutants with only the following mutations: E43T and E321V; The amino acid number is based on the amino acid sequence of the wild-type cysteine ​​convertase shown in SEQ ID NO:

1.

2. An isolated polynucleotide, characterized in that, The polynucleotide encodes the cysteine ​​convertase mutant of claim 1.

3. A carrier, characterized in that... The carrier contains the polynucleotide as described in claim 2.

4. A host cell, characterized in that, The host cell contains the vector of claim 3, or the polynucleotide of claim 2 is integrated into its genome; Furthermore, the host cell will not generate plant or animal individuals.

5. A method for preparing the cysteine ​​convertase mutant of claim 1, characterized in that, Including the following steps: (a) Under suitable expression conditions, the host cell of claim 4 is cultured to express the cysteine ​​convertase mutant of claim 1; (b) Isolate the expression product to obtain the cysteine ​​convertase mutant.

6. A method for lowering homocysteine ​​levels in vitro, characterized in that, Including the following steps: (a) In the presence of the cysteine ​​convertase mutant of claim 1, cysteine ​​is degraded in vitro to produce pyruvate. 。 7. A reaction system for the in vitro degradation of cysteine, characterized in that, The reaction system includes: (i) the cysteine ​​convertase mutant of claim 1; and (ii) Cysteine ​​substrate.

8. A pharmaceutical composition for treating colon cancer or glioblastoma, characterized in that, The pharmaceutical composition comprises: (i) pharmaceutically acceptable carriers; and (ii) The cysteine ​​convertase mutant as described in claim 1 or the polynucleotide as described in claim 2.

9. Use of a cysteine ​​convertase mutant as claimed in claim 1, a polynucleotide as claimed in claim 2, or a pharmaceutical composition as claimed in claim 8, characterized in that, Used to prepare pharmaceutical compositions for treating cancer; The cancer in question is colon cancer, glioblastoma, or a combination thereof.

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