Cysteine converting enzyme modification and application thereof

Abstract

The invention provides a separated or purified cysteine converting enzyme mutant and application thereof, and particularly relates to a medicine composition for preparing cancer treatment. In addition, the invention also provides a method for degrading cysteine in vitro, and the method comprises the step of degrading cysteine in vitro to generate pyruvic acid in the presence of the cysteine invertase mutant.

Description

Technical Field

[0001] The present invention belongs to the field of tumor treatment drugs, and in particular relates to the modification of cysteine ​​convertase and its application. Background Art

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

[0003] At present, metabolic therapy based on targeted degradation of specific amino acids that are highly required by tumor cells has gradually become an important means of tumor treatment. Among them, amino acid depletion therapy uses heterologous enzymes, recombinant enzymes or engineered enzymes to target and inhibit the uptake of specific amino acids and promote catabolism, which can effectively inhibit the growth of tumor cells. In addition, this therapy has much fewer side effects than chemotherapy and radiotherapy, and has little effect on normal cells.

[0004] As one of the few sulfur-containing amino acids and a component of glutathione, cysteine ​​is essential for the dynamic balance of redox in cells in addition to participating in protein synthesis and post-translational modification. When the intracellular content of cystine-cysteine ​​is depleted, the production of reduced glutathione (GSH), an important antioxidant substance in the cell, will be reduced, thereby seriously disrupting the intracellular redox balance and causing the cell to die. Tumor cells experience higher reactive oxygen species (ROS) oxidative stress than normal cells due to changes in cell genes and abnormal proliferation. Therefore, reduced glutathione (GSH), an intracellular antioxidant, is essential for cell survival and proliferation. Since cysteine ​​is one of the components of GSH, the increase in GSH may deplete endogenous sources of cysteine. Although tumor cells can produce cysteine ​​from methionine through homocysteine, this is often not enough to meet the demand. Therefore, tumor cells must take in a large amount of exogenous cysteine ​​in order to survive.

[0005] Therefore, there is an urgent need in the art 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 the present invention is to provide a mutant of bacterial-derived cysteine ​​(cysteine) convertase and use the mutant for preparing a drug for treating cancer.

[0007] In the first aspect of the present invention, an isolated or purified cysteine ​​invertase mutant is provided, wherein the mutant has the following site mutations: E43T, E321V, wherein the sites are based on the amino acid sequence of the wild-type cysteine ​​invertase 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: catalytic degradation of cysteine ​​in vitro.

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

[0011] In another preferred embodiment, the mutant-derived protein includes: an amino acid sequence formed by amino acid mutations E43T and E321V in the sequence shown in SEQ ID NO: 2, further having one or more amino acid residues deleted, inserted and / or substituted, and still having the activity of degrading cysteine ​​in vitro.

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

[0013] In the second aspect of the present invention, an isolated polynucleotide is provided, wherein the isolated polynucleotide encodes the cysteine ​​convertase mutant according to the first aspect of the present invention.

[0014] In another preferred embodiment, the polynucleotide is selected from the following group: DNA, RNA, cDNA or a combination thereof.

[0015] In the third aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide described in the second aspect of the present invention.

[0016] In another preferred embodiment, the vector includes bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

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

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

[0019] In the fourth aspect of the present invention, a host cell is provided, wherein the host cell contains the vector described in the third aspect of the present invention, or the polynucleotide described in the second aspect of the present invention is integrated into the genome of the host cell.

[0020] In another preferred embodiment, suitable host cells include Gram-positive bacteria, Gram-negative bacteria, actinomycetes, yeast and fungi, etc., wherein 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 yeast include but are not limited to Saccharomyces cerevisiae, and the fungi include but are not limited to Aspergillus, and their cells are all commonly used host cells for recombinant vectors.

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

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

[0023] In a fifth aspect of the present invention, there is provided a method for preparing the cysteine ​​convertase mutant according to the first aspect of the present invention, comprising the steps of:

[0024] (i) culturing the host cell according to the fourth aspect of the present invention under conditions suitable for expression, thereby expressing the (cysteine) convertase mutant according to the first aspect of the present invention;

[0025] (ii) isolating the expression product, thereby obtaining the cysteine ​​(cysteine) convertase mutant.

[0026] In a sixth aspect of the present invention, there is provided a method for degrading (half) cysteine ​​in vitro, comprising the steps of:

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

[0028]

[0029] In another preferred embodiment, the in vitro method for degrading (half) cysteine ​​is an in vitro method for degrading (half) cysteine ​​for non-diagnostic and non-therapeutic purposes.

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

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

[0032] (i) the cysteine ​​convertase mutant according to the first aspect of the present invention; and

[0033] (ii) Cysteine ​​substrate.

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

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

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

[0037] (ii) the cysteine ​​(cysteine) convertase mutant according to the first aspect of the present invention or the polynucleotide according to the second aspect of the present invention.

[0038] In the ninth aspect of the present invention, there is provided a use of the cysteine ​​convertase mutant described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention or the pharmaceutical composition described in the eighth aspect of the present invention, which is used to prepare 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 liquid cancer.

[0042] In another preferred embodiment, the liquid cancer includes non-Hodgkin's lymphoma (NHL), acute lymphocytic 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 the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0045] Figure 2 The BacCyse expression recombinant plasmid is shown.

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

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

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

[0049] Figure 4 It was shown that the BacCyse protein is able to degrade cysteine.

[0050] Figure 4 A shows the OD values ​​of the experimental group and the NT group after incubation at 37°C for 2 hours with 2.5 mM cysteine ​​as substrate and 1 μg / mL BacCyse protein. 600 . Figure 4 B shows the OD values ​​detected at different time points of 30 min, 2 h, 8 h, 32 h, and 56 h when 2 mM cysteine ​​was used as substrate and 10 μg / mL BacCyse protein was added. The control group was added with an equal amount of PBS. 600 .

[0051] Figure 5 It shows that Cyst(e)inase protein has stronger cysteine ​​decomposition ability than MccB protein.

[0052] Figure 5 A shows the remaining cysteine ​​concentrations of 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 degrading 1 mM cysteine ​​standard at 37°C for 2 h.

[0053] Figure 6 It was shown that BacCyse protein inhibited the growth of HCT116 cells.

[0054] Figure 6 A shows that the starting number of HCT116 cells per well is 7*10 4After 72 hours of culture at 37°C and 5% carbon dioxide, the cells were photographed and observed. The NT group was added with the corresponding amount of PBS; Figure 6 B shows the ratio of the number of cells in the control group (NT) at a concentration of 1 μg / mL cysteine ​​amino acidase after 72 hours. Three biological replicates were performed, and each biological replicate in each group had three replicates. ***, p<0.001.

[0055] Figure 7 It was shown that BacCyse protein could inhibit the growth of LN229 cells.

[0056] Figure 7 A shows that the starting number of LN229 cells per well was 7.5*10 4 , cultured at 37°C and 5% carbon dioxide for 24, 48, and 72 hours, and then photographed and observed the cell conditions. The NT group was added with a corresponding amount of PBS, and the drug group was added with 10ug / ml of BacCyse protein; Figure 7 B shows the percentage of cells in the BacCyse protein-treated group compared with the control group (NT) after 24 hours, 48 ​​hours, and 72 hours; Figure 7 C shows the changes in the number of cells in the BacCyse protein treatment group and the control group (NT) after 24 hours, 48 ​​hours, and 72 hours. FM-control group, complete medium. FM-(cysteine)ase is complete medium with 10ug / ml BacCyse protein added. Three biological replicates were performed, and each biological replicate in each group had three replicates.

[0057] Figure 8 It was shown that BacCyse protein inhibited the growth of MC38 cells.

[0058] Figure 8 A shows that the starting number of MC38 cells per well is 8*10 4 After culturing at 37°C and 5% carbon dioxide for 24, 48 and 72 hours, the cells were photographed and observed. The NT group was added with a corresponding amount of PBS, and the (cysteine) enzyme group was added with 10ug / ml BacCyse protein. Figure 8 B shows the ratio of the number of cells in the BacCyse protein-treated group compared to the control group (NT) after 24 hours, 48 ​​hours, and 72 hours. Figure 8 C shows the changes in the number of cells in the BacCyse protein-treated group and the control group (NT) after 24 hours, 48 ​​hours, and 72 hours. FM-control group, complete medium. FM-(cysteine)ase is complete medium with BacCyse protein added. Three biological replicates were performed, and each biological replicate in each group had three replicates.

[0059] Fig. 9 The schematic diagram of in vivo treatment of mouse tumor-bearing model is shown. 6-8w C57 / 6j mice were injected with MC38-1*10 6 The NT group was intraperitoneally injected with corresponding amount of PBS on the seventh, ninth, eleventh and fourteenth days after subcutaneous tumor formation. The experimental group was intraperitoneally injected with BacCyse protein on the seventh, ninth, eleventh and fourteenth days after subcutaneous tumor formation, and the IR group received radiotherapy on the ninth day after subcutaneous tumor formation.

[0060] Fig.10 The results show that BacCyse protein inhibits tumor growth in mice. NT (n=5), IR (10 Gray; n=6), cysteinease (100 mg / kg BacCyse protein; n=5). *, p<0.05. DETAILED DESCRIPTION

[0061] The inventors have conducted extensive and in-depth research, and through a large number of screenings and studies on mutant proteins, unexpectedly obtained a mutant of bacterial cysteine ​​convertase (BacCyse, Bacterial L-cyst(e)inase) for the first time. It was found that the mutant of cysteine ​​convertase can efficiently degrade extracellular cysteine, thereby reducing the extracellular cysteine ​​concentration of tumor cells and effectively inhibiting the growth and proliferation of various cancer cells. The present invention was completed on this basis.

[0062] Specifically, the present invention clones the natural mccB gene from Bacillus subtilis, which is commonly found in food and the environment, and performs site-specific processing and modification on its protein sequence, mutating the glutamic acid at position 43 to threonine (E43T), and mutating the glutamic acid at position 321 to valine (E321V), and finally obtaining a new (cysteine) hydrolase, named BacCyse.

[0063] The activity of the mutated cysteine ​​convertase in degrading cysteine ​​is increased by about 5 times, and it can also directly degrade pyruvate. The cysteine ​​convertase mutant of the present invention can effectively inhibit the growth of mouse tumors, and the effect of the initial administration is equivalent to that of irradiation.

[0064] As used herein, "(cysteine) convertase", "wild-type (cysteine) convertase", "cysteine ​​convertase", "wild-type cysteine ​​convertase" or "wild-type MccB protein" are used interchangeably to refer to (cysteine) convertase. It should be understood that the (cysteine) convertase refers to a convertase of cysteine ​​or cystine.

[0065] In the present invention, "mutant of (half) cysteine ​​convertase", "mutated (half) cysteine ​​convertase", "cysteine ​​convertase mutant", "mutant", "mutant protein", "active polypeptide of the present invention", "(half) cysteine ​​enzyme", "Cyst(e)inase" or "BacCyse protein" can be used interchangeably to refer to the (half) cysteine ​​convertase mutant described in the first aspect of the present invention.

[0066] Existing engineered enzymes for reducing cysteine ​​mainly come from eukaryotic organisms, but the eukaryotic protein expression system has disadvantages such as many procedures and high cost. In addition, the codons of eukaryotic proteins are often rare in prokaryotes, resulting in low yields and easy formation of inclusion bodies in the lower-cost Escherichia coli expression system.

[0067] The present invention is a cysteine ​​convertase derived from prokaryotes, the enzyme sequence length of which is only 379 amino acids, which is easy to express and purify on a large scale in engineered bacteria, and is suitable for industrial fermentation and production. At the same time, the mutant obtained by the present invention has better in vitro cysteine ​​degradation activity and strong ability to inhibit cancer cell growth.

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

[0069] The wild-type MccB protein of bacteria has cystathionine-lyase and homocysteine ​​γ-hydrolase activities in vitro. MccB has 50% similarity with cystathionine β-hydrolase (MetC) and 46% similarity with cystathionine γ-hydrolase of rat and Saccharomyces cerevisiae. The activity of the purified MccB enzyme obeys Michaelis-Menten kinetics, with a Km value of about 3mM, and generates about 2μmol of free thiol groups per mg of protein per minute (V max ). The degradation of cysteine ​​by cysteine ​​γ-hydrolase produces free thiol-containing compounds, namely homocysteine ​​or cysteine.

[0070] Tumor cells can accelerate the uptake of extracellular cystine by upregulating the cystine / glutamate antiporter (xCT transporter). Figure 1 As shown, after the cystine taken up from outside the cell reaches the cell, part of it will be converted into cysteine, thereby increasing the synthesis of GSH.

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

[0072] In a specific embodiment, the wild-type (cysteine) invertase and the gene encoding the same 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 ​​invertase 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 derivative protein, which has amino acids E43 and E321, and has ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99% sequence identity compared to the wild-type (cysteine) convertase, and has the activity of degrading cysteine ​​in vitro.

[0076] In a specific embodiment, the derivative protein comprises: an amino acid sequence formed by amino acids E43 and E321 in the sequence shown in SEQ ID NO: 1, further having one or several (for example, usually 1-30, preferably 1-10, more preferably 1-6, still more preferably 1-3, and most preferably 1) amino acid residues deleted, inserted and / or substituted, and still has the activity of degrading cysteine ​​in vitro.

[0077] In a specific embodiment, the mutant of the cysteine ​​invertase is a mutant of the cysteine ​​invertase having the following mutations: E43T, E321V, wherein the sites are based on the amino acid sequence of the wild-type cysteine ​​invertase shown in SEQ ID NO:1.

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

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

[0080] In a specific embodiment, the mutant can degrade exogenous cysteine ​​in tumor cells in vitro, thereby inhibiting the growth of tumor cells, and the activity is significantly improved compared with the wild type.

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

[0082] MKKKTLMIHGGITGDEKTGAVSVPIYQVSTYKQPKAGQHTGY TYSRTANPTRTALEALVTELESGEAGYAFSSGMAAITAVMMLFNSGDHVVLTDDVYGGTYRVMTKVLNRLGIESTFVDTSSREEVEKAIRPNTKAIYIETPTNPLLKITDLTLMADIAKKAGVLLIVDNTFNTPYFQQP LTLGADIVLHSATKYLGGHSDVVGGLVVTASKELGEELHFVQNSTGGVLGPQDSWLLMRGIKTLGLRMEAIDQNARKIASFLENHPAVQTLYYPGSSNHPGHELAKTQGAGFGGMISFDIGSEERVDAFLGNLKLFTIA V SLGAVESLISVPARMTHASIPRERRLELGITDGLIRISVGIEDAEDLLEDIGQALENI*.

[0083] In a specific embodiment, the mutant protein of the present invention also includes fragments, derivatives and analogs of the mutant shown in SEQ ID NO: 2, wherein the fragments, derivatives and analogs substantially maintain the activity of the (half) cysteine ​​convertase mutant of the present invention, which may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) a polypeptide having a substitution group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of the polypeptide of the present invention with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence (a fusion protein formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as His). These fragments, derivatives and analogs belong to the scope known to those skilled in the art.

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

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

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

[0087] In a specific embodiment, analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as β, 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 a specific embodiment, the amino or carboxyl terminus of the protein of the present invention may also 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 to purify the protein.

[0089] In a specific embodiment, in order to secrete the translated protein (e.g., secrete it outside the cell), a signal peptide sequence, such as a pelB signal peptide, may be added to the amino acid amino terminus of the (cysteine) convertase mutant. The signal peptide may be cleaved during the process of secretion of the polypeptide from the cell.

[0090] The present invention relates to a polynucleotide encoding the mutant of the present invention.

[0091] In a specific embodiment, the polynucleotide 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 for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

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

[0094] In a specific embodiment, the present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention. The variants of this polynucleotide can be naturally occurring variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the polypeptide encoded by it.

[0095] In a specific embodiment, the present invention also relates to a polynucleotide that hybridizes with the above sequence and has at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to a polynucleotide that can hybridize with the polynucleotide of the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. In addition, 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 a specific embodiment, the present invention also relates to a nucleic acid fragment that hybridizes with the above sequence. As used herein, a "nucleic acid fragment" has a length of at least 15 nucleotides, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides. The nucleic acid fragment can be used in nucleic acid amplification techniques (such as PCR) to identify and / or isolate polynucleotides encoding (cysteine) enzyme mutants.

[0097] In a specific embodiment, the polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably purified to homogeneity. The full-length sequence of the (half) cysteinease mutant of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0098] In a specific embodiment, the relevant sequence can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0099] In a specific embodiment, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis. The method of amplifying DNA / RNA using PCR technology is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used. The primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

[0100] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the coding sequence of the (cysteine) enzyme mutant, and a method for producing the mutant of the present invention by recombinant technology.

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

[0102] In a specific embodiment, the polynucleotide sequence of the cysteinease mutant can be inserted into a recombinant expression vector. The "recombinant expression vector" refers to a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus such as an adenovirus, a retrovirus or other vector well known in the art. As long as it can replicate and be stable in the host, any plasmid or vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0103] In a specific embodiment, a person skilled in the art can construct an expression vector containing a (half) cysteinease mutant encoding DNA sequence and appropriate transcription / translation control signals using well-known methods. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: lac or trp promoters of Escherichia coli; lambda phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retroviral LTRs and other known promoters that can 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 selectable marker genes to provide a phenotypic trait for selecting transformed host cells, 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 embodiment, a vector comprising the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the protein.

[0106] In a specific embodiment, the host cell can be a prokaryotic cell, such as a bacterial cell; or 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; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells, etc.

[0107] In a specific embodiment, 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, all of which are commonly used host cells for recombinant vectors.

[0108] In a specific embodiment, when the polynucleotide of the present invention is expressed in a higher eukaryotic cell, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, which act on the promoter to enhance the transcription of the gene. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0109] In a specific embodiment, those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0110] In a specific embodiment, the transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with CaCl 2 Another method is to use MgCl 2 If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

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

[0112] In a specific embodiment, the recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using 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 renaturation treatment, treatment with protein precipitants (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

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

[0114] As used herein, the cysteine ​​substrate includes a member selected from homocystine, cystine, cysteine ​​or a combination thereof.

[0115] application

[0116] The present invention also provides a pharmaceutical composition, comprising (i) a pharmaceutically acceptable carrier and (ii) the cysteinease mutant according to the first aspect of the present invention or the polynucleotide according to the second aspect of the present invention.

[0117] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation or other materials well known in the art for pharmaceutical preparations. It should be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration in a specific application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the effectiveness of the composition according to the present invention or the biological activity of the composition according to the present invention.

[0118] In a specific embodiment, the pharmaceutical composition can be in any suitable form, depending on the method of administration required by the patient. It can be provided in the form of a unit dosage form, usually placed in a sealed container, and can be provided as part of a kit. Such a kit usually (but not necessarily) includes instructions for use. It can include a plurality of the unit dosage forms.

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

[0120] The present invention also provides the use of the cysteinease mutant of the present invention, the polynucleotide of the present invention and the pharmaceutical composition of the present invention for preparing a pharmaceutical composition for treating cancerous rash.

[0121] The cancer can be any liquid cancer or solid cancer, for example, including but not limited to lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, 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 lymphocytic 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 separated, produced or purified from other biological components naturally occurring in the organism, including other chromosomal and extrachromosomal DNA and RNA and proteins. Thus, "isolated" nucleic acids, peptides and proteins include nucleic acids and proteins purified by standard purification methods. "Isolated" nucleic acids, peptides, proteins can become part of a complex, and if the complex is not part of the natural environment of the nucleic acid, peptide or protein, the nucleic acid, peptide, protein is still isolated. The term also includes nucleic acids, peptides and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acids.

[0123] As used herein, the term "peptide", "polypeptide" or "protein" can be a molecule composed of amino acids, which can be regarded as a protein by a person skilled in the art. The present invention uses conventional coding methods for amino acid residues, i.e., one-letter or three-letter codes. The terms "peptide", "polypeptide" and "protein" can be used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and it can be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified naturally or by intervention; the modification method is, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification, such as binding to a marker, etc. The definition of the term also covers, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art.

[0124] Modified forms (usually without changing the primary structure) include: chemical derivatization forms of polypeptides in vivo or in vitro, such as acetylation or carboxylation. Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine), and polypeptide forms modified to improve their anti-proteolytic properties or optimize their solubility properties.

[0125] The peptide sequences described in the present invention are written according to the usual convention, i.e. the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although isomeric forms of amino acids are known, unless otherwise expressly indicated, the L-form of the amino acid is described.

[0126] In a specific embodiment, for example, in the art, when an amino acid with similar or similar properties is substituted, the function of the protein is usually not changed. For another example, adding one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein.

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

[0128] Advantages of the present invention

[0129] 1. The present invention obtains the cysteine ​​convertase mutant of the present invention through a large number of screening and directed mutagenesis, and unexpectedly finds that the obtained cysteine ​​convertase mutant can efficiently degrade cysteine ​​in vitro, and the enzyme activity of the mutant is about five times that of the wild-type cysteine ​​convertase.

[0130] 2. The cysteine ​​convertase mutant of the present invention can significantly inhibit the proliferation of tumor cells.

[0131] 3. The cysteine ​​convertase mutant of the present invention can significantly inhibit tumor growth.

[0132] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually 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 under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts. Specific embodiments

[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 carcinoma 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. The MC38 culture medium is DMEM culture medium containing 10% FBS and 1% penicillin-streptomycin. The culture conditions are 37°C and 5% CO2 saturated humidity.

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

[0143] Human colorectal cancer cells-HCT116 are mainly used for in vitro cell proliferation experiments. HCT116 culture medium is McCoy's 5A culture medium containing 10% FBS and 1% penicillin-streptomycin. The culture conditions are 37°C and 5% CO2 saturated humidity.

[0144] c) LN229 neuroblastoma cell line culture

[0145] Human neuroblastoma cell-LN229 is mainly used for in vitro cell proliferation experiments. LN229 culture medium is DMEM culture medium containing 10% FBS and 1% penicillin-streptomycin. The culture conditions are 37°C and 5% CO2 saturated humidity.

[0146] d) Mouse tumor model

[0147] Wild mice with C57BL / 6J genetic background of 6-8 weeks old were selected. After anesthetizing the mice by intraperitoneal injection of avertin anesthetic, the right thigh of the mice was shaved cleanly with a razor to expose the epidermis. A 1*10 6 MC38 mouse colon cancer cells were subcutaneously inoculated. To prevent the injection solution from leaking out, the needle was carefully pulled out 3 seconds after the injection was completed.

[0148] e) Mouse treatment model

[0149] After the mouse model was successfully established by subcutaneous injection of MC38, the size of the subcutaneous tumor in the mouse reached 100-200 mm 3 During the period between 2-3 days, the tumor-bearing mice were randomly divided into groups and given different doses of protein drugs by intraperitoneal injection using a 1 mL syringe, and the tumors were subsequently measured every 2-3 days. According to needs, some tumor-bearing mice were irradiated and then given drugs to evaluate the effect of combined treatment.

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

[0151] (f1) Transform the BacCyse protein recombinant plasmid into BL21 (DE3) competent E. coli, incubate on ice for 30 minutes, place in a 42°C water bath for 1 minute, and then quickly place on ice for 2-5 minutes. Add 1 mL of LB medium (without antibiotics) to the competent cells and incubate at 37°C, 220 rpm, for 1 hour. Plate and incubate at 37°C for 16 hours.

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

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

[0154] (f4) Add 15 mL of the bacterial solution in the shaking tube to 1 L of LB medium (containing kanamycin resistance) at 37°C, 220 rpm, for 2-3 hours. Induce when OD600 is 0.6.

[0155] (f5) Take 1 mL and store it in a 1.5 mL EP tube as a pre-induction control. Add IPTG to the rest of the bacterial solution to induce at a final concentration of 1 mM, 25°C, 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) The induced bacterial solution was centrifuged at 6000 rpm for 10 minutes, 40 mL of lysis buffer and protease inhibitors were added, and ultrasonic disruption was performed at 40% duty, 5s on, 3s off, for 30 minutes. After disruption, the solution was centrifuged at 17000 rpm for 30 minutes. The supernatant was collected and filtered with a 0.45 um filter membrane. The filtered liquid was used for the next experiment.

[0157] (f7) Ni-NTA column: mix the supernatant with the Ni column and mix at 4°C for 1 hour. Place the column vertically for 10 minutes, collect the effluent, and resuspend the Ni column with 1 column volume of the protein eluent. Repeat two or three times, and then wash the column with a total of 10 column volumes of the protein eluent.

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

[0159] (f9) The collected target protein eluate was placed in an activated dialysis bag, which was then placed in pre-cooled PBS to replace the buffer at 4°C for 24 hours. Finally, the target protein was subjected to SDS-PAGE detection and concentration determination.

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

[0161] (g1) All items were removed without heat source, and the container was soaked in 0.5M NaOH for 30 minutes and then washed with heat-free ultrapure water.

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

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

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

[0165] h) Statistical analysis

[0166] Quantitative data in the present invention are expressed as mean + standard error (SEM). Significant differences were tested by T-test. When the P value was less than 0.05, it was considered statistically significant. The data were derived from more than three independent experiments, and at least 3 replicates were set for each independent experiment.

[0167] Example 1 Construction of BacCyse protein expression plasmid

[0168] The BacCyse expression recombinant plasmid was constructed based on the pET-28a plasmid vector. Figure 2 As shown in A. The recombinant plasmid was digested with single enzyme (BamH I) and double enzyme (BamH I, Xho I) respectively, and then analyzed by agarose gel electrophoresis.

[0169] Agarose gel electrophoresis results Figure 2 As shown in B, the figure shows a single band of about 6500 bp (single enzyme digestion) and double bands of about 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 the recombinant plasmid was transformed into E. coli, BacCyse protein was expressed and purified. The specific steps were: after induction at 37°C with 1 mM IPTG for 16 hours, the bacteria were collected by centrifugation, and the supernatant was collected after ultrasonic disruption; SDS-PAGE electrophoresis was followed by Coomassie Brilliant Blue staining. Figure 3 A shows a large amount of target protein expression. Subsequently, the BacCyse protein with the His tag was purified using a Ni column; the protein dissolution buffer was replaced with a phosphate buffer through a dialysis bag, and the BacCyse protein was finally obtained. A small amount of protein was taken for SDS-PAGE gel running and stained with Coomassie Brilliant Blue. The results are as follows Figure 3 As shown in B.

[0172] Depend on Figure 3 C and Figure 3 Comparison results with the protein standard BSA in D show that the purity of BacCyse protein is about 93% and the concentration is about 2.5 mg / mL.

[0173] Example 3 Compared with the wild-type protein, the enzymatic activity of BacCyse protein in degrading cysteine ​​is higher

[0174] Cysteine ​​contains sulfhydryl groups, which can reduce phosphate to generate tungsten blue. 600 There is a maximum absorption peak, so by detecting OD 600 The absorbance reflects the reduction of cysteine ​​to phosphorylation and indirectly detects the degradation of cysteine ​​by BacCyse protein.

[0175] The results are as follows Figure 4 As shown in 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 ​​substrates.

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

[0177] In order to explore the activity of MccB protein before mutation and BacCyse protein after mutation in degrading cysteine, BacCyse protein and MccB protein with experimental concentration of 10ug / mL were reacted with 1mM cysteine ​​standard for 2h at 37℃, and the absorbance value was analyzed using cysteine ​​content detection kit. The control group was treated at 95℃ for 5 minutes.

[0178] Figure 5 A and Figure 5 The results of B showed that both MccB protein and BacCyse protein 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 ​​decomposition ability than MccB protein, and the enzyme activity efficiency is about 5 times that of MccB protein.

[0179] After heat inactivation, the ability of MccB and BacCyse proteins to degrade cysteine ​​disappeared.

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

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

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

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

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

[0185] The BacCyse protein was tested to see if it could play a role in the mouse colon cancer MC38 cell line. After 24 hours, 48 ​​hours, and 72 hours of BacCyse protein treatment, the cell growth was as follows: Figure 8 As shown in A.

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

[0187] The data of this example 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 can significantly inhibit the growth of MC38 tumors in tumor-bearing mice

[0189] Based on MC38 tumor-bearing mice, we tested whether BacCyse protein could inhibit tumor growth. First, we selected C57 / 6j mice with a clear genetic background, and inoculated MC38 colon cancer tumor cell lines into the right leg of 6-8w mice to construct a subcutaneous tumor-bearing model. Fig. 9 As shown, cysteine ​​amino acid protein was injected intraperitoneally starting from the seventh day of tumor-bearing mice, with injections every two to three days, for a total of four doses; in order to better reflect the therapeutic effect of cysteine ​​amino acid protein in the mouse tumor model, radiotherapy, a mature tumor treatment method currently used in clinical practice, was selected as a positive control experiment, and radiotherapy was performed once on the ninth day of tumor-bearing mice.

[0190] The results are as follows Fig.10 As shown, by continuously measuring tumor size, it was proved that BacCyse protein alone in vivo can significantly inhibit tumor growth; within two days after the last injection of BacCyse protein, the inhibitory effect on tumors was equivalent to that of radiotherapy, and then the effect was slightly weaker than radiotherapy, but there was still a significant difference compared with the control group.

[0191] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. An isolated or purified cysteine ​​convertase mutant, characterized in that The mutant is a cysteine ​​convertase mutant having the following mutations: E43T, E321V; The amino acid numbering is based on the amino acid sequence of the wild-type cysteine ​​invertase shown in SEQ ID NO:

1.

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

3. A carrier, characterized in that The vector contains the polynucleotide according to claim 2.

4. A host cell, characterized in that The host cell contains the vector according to claim 3, or the polynucleotide according to claim 2 is integrated into its genome.

5. A method for preparing the cysteine ​​convertase mutant according to claim 1, characterized in that: Includes steps: (a) culturing the host cell according to claim 4 under conditions suitable for expression, thereby expressing the cysteine ​​convertase mutant according to claim 1; (b) isolating the expression product to obtain the cysteine ​​convertase mutant.

6. A method for reducing cysteine ​​in vitro, characterized in that: Includes steps: (a) in the presence of the cysteine ​​convertase mutant according to claim 1, degrading cysteine ​​in vitro to generate pyruvate 7. A reaction system for degrading cysteine ​​in vitro, characterized in that: The reaction system comprises: (i) the cysteine ​​convertase mutant according to claim 1; and (ii) Cysteine ​​substrate.

8. The reaction system according to claim 7, characterized in that The cysteine ​​substrate is selected from the group consisting of cysteine, cystine, homocystine, or a combination thereof.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (i) a pharmaceutically acceptable carrier; and (ii) the cysteine ​​convertase mutant according to claim 1 or the polynucleotide according to claim 2.

10. A use of the cysteine ​​convertase mutant according to claim 1, the polynucleotide according to claim 2 or the pharmaceutical composition according to claim 9, characterized in that: Used for preparing a pharmaceutical composition for treating cancer.

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