Arginine-free protein and use thereof

Abstract

The application discloses a kind of protein without arginine and application thereof.A group of high-score proteins without or with only very low amount of arginine is obtained by designing and modifying amino acid sequences.The high-quality protein powder for food is finally obtained by optimizing microbial fermentation and purification process.The protein powder prepared in the application has high purity and is easy to absorb, and the content of arginine is less than 1.0g / 100g protein.The content of other amino acids except arginine is rich and balanced.The product of the application can be used to reduce or control the level of arginine in vivo, and treat arginemia, tumor and arthritis, etc.The product of the application can replace natural protein as protein supplement for specific population.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to an arginine-free protein and its applications. Background Technology

[0002] Arginine is a non-essential amino acid and an important precursor in the production of proteins, polyamines, creatinine, and nitric oxide (NO). In chronic diseases, arginine levels affect systemic NO levels, playing a pro-inflammatory role and increasing the risk of cancer. Therefore, limiting arginine intake may improve certain pathological conditions and enhance disease treatment outcomes. After more than a decade of research, strategies for limiting arginine intake have evolved into two main directions: inhibiting arginine transport and breaking down arginine in the body. Methods for breaking down arginine mainly include limiting dietary arginine intake or degrading arginine in the body through proteases.

[0003] Currently, the only method for restricting dietary arginine intake is to use dietary amino acid blends. Arginine is removed from the blend, and the resulting amino acid powder replaces the daily protein intake for patients. This method is used to treat various diseases. For example, dietary arginine restriction is the primary clinical treatment for argininemia. Argininemia, also known as arginase deficiency, is caused by a lack of the enzyme that hydrolyzes arginine, resulting in significantly elevated arginine levels in the blood and cerebrospinal fluid. Clinical symptoms include intellectual and motor impairments, spastic paralysis, etc. Besides argininemia, dietary arginine restriction has also shown strong therapeutic potential in animal tumor models. For instance, a study by Yeatman et al. (ArchSurg 1991; 126: 1376-81) showed that arginine supplementation increased the growth of metastatic colon tumors in mice by 55%, while dietary arginine restriction inhibited tumor growth by up to 78%. Furthermore, a recent study by Brunner et al. (Nat Commun. 2020;11(1):431) showed that the consumption of the amino acid arginine inhibits osteoclast formation and improves arthritis in mice. These studies suggest that limiting arginine intake can have therapeutic effects on various diseases. However, the poor taste of amino acid compound powders leads to poor patient compliance, limiting clinical efficacy compared to animal studies. Unlike proteins derived from food, this free amino acid mixture does not require digestion by gastrointestinal proteases, resulting in faster absorption but also faster excretion. Absorption kinetics studies show that after taking the free amino acid mixture, the total amino acid level in plasma rises and falls rapidly, significantly different from the relatively gentle absorption kinetics curve of food proteins (PEN J ParenterEnteral 1991.15: 48-53). This rapid fluctuation in plasma amino acid levels means that the rate of new protein synthesis cannot keep up with the rate of amino acid loss, leading to a significant increase in nitrogen loss and a decrease in assimilation efficiency (Gut 1983. 24: 78-84).

[0004] Besides dietary restrictions, arginine-depleting enzymes are under clinical development. One type is PEGylated recombinant human arginase 1 (AGR1), including BCT-100 and Pegzilarginase, which degrades arginine to produce ornithine and urea; another type is PEGylated bacterial arginine deiminase (ADI-PEG 20), which breaks down arginine to produce citrulline and ammonia. ADI-PEG 20 and BCT-100 are mainly used to treat malignant tumors, while pegzilarginase is in Phase 3 clinical development for the treatment of ARG1 deficiency (NCT03921541). Clinical data show that these protease drugs, after intravenous or intramuscular injection, can significantly reduce the level of arginine in the human blood in a short period of time, and long-term use alone or in combination with other anti-tumor drugs has considerable efficacy for patients with advanced solid tumors. However, these drugs have some significant problems. For example, antibodies can be detected in most patients 2-4 weeks after injection of ADI-PEG 20, causing the effect to be lost at low doses. BCT-100 and Pegzilarginase, developed based on human arginase 1 (AGR1), have significantly reduced immunogenicity. However, for cancer patients, some tumor cells can synthesize arginine using ornithine, a degradation product of AGR1, through a salvage pathway. Therefore, BCT-100 and Pegzilarginase have poor efficacy in treating tumors.

[0005] In summary, arginine restriction shows promising applications in the treatment of cancer, inflammatory diseases, and argininemia. However, current methods for arginine restriction have limitations. Therefore, it is necessary to research and develop better arginine restriction strategies. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a recombinant dietary protein and its composition that is free of arginine and has high biological value. Consuming this protein and its composition can effectively reduce or control arginine levels in the body, while simultaneously supplementing other essential amino acids and nutrients. This protein and its composition can be used to inhibit tumor growth, treat hyperarginemia and arthritis, and in any product application field aimed at reducing arginine intake and promoting arginine consumption in the body.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, a recombinant dietary protein without arginine is provided, comprising a polypeptide sequence having at least 70% identity with the amino acid sequence shown in any one of SEQ ID NO. 1-12.

[0008] In one embodiment, the recombinant protein comprises at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO. 1-12, with increasing priority.

[0009] In a preferred embodiment, the recombinant dietary protein comprises a polypeptide sequence having 100% identity with the amino acid sequence shown in any one of SEQ ID NO. 1-12.

[0010] In a more preferred embodiment, the recombinant dietary protein comprises the amino acid sequence shown in SEQ ID NO. 1 or 2; in an even more preferred embodiment, the recombinant dietary protein comprises the amino acid sequence shown in SEQ ID NO. 1.

[0011] In a second aspect, a vector is provided that contains a nucleic acid sequence encoding the recombinant dietary protein described in the first aspect.

[0012] Thirdly, a recombinant microorganism is provided, comprising the vector provided in the second aspect.

[0013] Preferably, the microorganisms include, but are not limited to, any one of the following expression systems: *Escherichia coli*, filamentous fungi, *Bacillus*, and yeast. Further, the *Escherichia coli* expression system is the *Escherichia coli* K12 expression system; the filamentous fungi expression system is the *Trichoderma reesei* expression system; the *Bacillus* expression system is the *Bacillus licheniformis* or *Bacillus subtilis* expression system; and the yeast expression system is the *Pichia pastoris* expression system. Even more preferably, the *Bacillus licheniformis* strain is 10266Δ9.

[0014] Fourthly, a method for preparing the recombinant dietary protein described in the first aspect is provided, the method comprising culturing the recombinant microorganism of the third aspect under conditions suitable for producing dietary protein by said recombinant microorganism.

[0015] Fifthly, a composition containing recombinant protein, wherein each 1 g of total protein in the composition contains no more than 10 mg of arginine.

[0016] Preferably, the main protein component of the composition is a recombinant dietary protein, wherein the recombinant dietary protein is free of arginine or contains no more than 1.0% arginine.

[0017] More preferably, the main protein component of the composition is the recombinant dietary protein described in the first aspect.

[0018] Sixthly, the use of the recombinant dietary protein described in the first aspect and the composition described in the fifth aspect in the preparation of products that reduce the intake and consumption of arginine in the body is provided.

[0019] In a seventh aspect, the recombinant dietary protein described in the first aspect and the composition described in the fifth aspect are provided for use in the preparation of medicaments or special medical foods for controlling and treating argininemia, arthritis, or inhibiting tumor growth.

[0020] As used in this invention, “recombinant” refers to a biomolecule, such as a gene or protein, that: (1) has been removed from its naturally occurring environment; (2) is wholly or partially unrelated to a polynucleotide in a naturally occurring gene; (3) is operatively linked to a polynucleotide not linked to in nature; and / or (4) is not present in nature. The term “recombinant” can also refer to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biosynthesized by a heterologous system, as well as proteins and / or mRNA encoded by these nucleic acids. Thus, for example, a protein synthesized by a microorganism is recombinant if it is synthesized from mRNA synthesized from a recombinant gene present in the cell.

[0021] As used herein, the term "dietary protein" refers to protein suitable for human consumption. Dietary proteins that provide all essential amino acids are considered to have high biological value. Proteins that provide all other essential amino acids but are arginine-free are also considered to have high biological value. Casein (a protein commonly found in mammalian milk, comprising 80% of the protein in milk) and whey (the protein remaining in the liquid after milk has been coagulated and filtered) are major sources of dietary proteins with high biological value. Except for Met, the dietary proteins of this invention contain all essential amino acids.

[0022] The term "essential amino acids," as described in this invention, refers to histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), threonine (Thr, T), tryptophan (Trp, W), and valine (Val, V), which are amino acids required for health and growth but cannot be synthesized by the human body and must be obtained from food.

[0023] The term "recombinant microorganism" as described in this invention refers to a microorganism modified to carry a copy of a recombinant gene.

[0024] As used in this invention, the term "naturally occurring protein" refers to a protein derived from a sequence present in a natural host that has not been artificially altered. Therefore, neither the DNA sequence encoding the protein nor the amino acid sequence of the protein itself exhibits any sequence changes found in the natural host.

[0025] The term "foods for special medical purposes" refers to foods for special medical purposes (FSMP), also known as medical foods. These are specially processed and formulated foods designed to meet the specific nutritional or dietary needs of individuals with restricted food intake, digestive and absorption disorders, metabolic disorders, or specific disease states. They are novel products distinct from ordinary foods, health supplements, and medicines, and are a category of foods with special dietary uses, taken under the guidance of a doctor by patients requiring special dietary management.

[0026] The beneficial effects of this invention are: This invention designs and optimizes a high-scoring protein that is arginine-free but contains other essential amino acids. The resulting protein powder, obtained through efficient expression in a safety-certified microbial system, has an arginine content ≤ 10.0 mg / g of protein. The protein is inactivated in vitro to eliminate its functionality, but retains its ability to be rapidly degraded by gastrointestinal proteases, making it easily digestible in vivo. It also exhibits the same absorption pattern as natural proteins, with virtually no immunogenicity or toxic side effects. The production process is simple, easy to operate, and low-cost, allowing for large-scale factory production and providing a long-term, cost-effective product for those in need. Animal experiments have verified that feeding patients with arginine-free recombinant dietary protein and its compositions as a substitute for dietary protein can effectively reduce blood arginine levels. This invention can be used to inhibit tumor growth and treat hyperarginemia and arthritis. Attached Figure Description

[0027] Figure 1 SDS-PAGE of ArgR1-1, ArgR2-1 and ArgR4-1.

[0028] Figure 2 Mouse breast cancer xenograft volume ( p <0.001).

[0029] Figure 3 Statistical analysis of food intake in mice with breast cancer xenografts.

[0030] Figure 4 Body weight of mice with breast cancer xenografts on day 0 and day 40.

[0031] Figure 5 Weight statistics of mice in an arthritis model. Detailed Implementation

[0032] Example 1: Design and expression of Arginine-free ArgR1 series proteins This example uses genetically modified Bacillus licheniformis to express ArgR1 series recombinant proteins without arginine.

[0033] The ArgR1 series protein variants share the same protein backbone, and their mature peptide sequences are as follows: AQX1VPYGX2PLIKAX3KX4QX5QGFKGX6NVKVAVX7DX8GIX9X10SHPDLNVX11GGASX12VX13X14EX15X16X17X18DGNGHGTHVAG TVAALX19NX20TGVLGVAPSX21SLYAVKVLX22X23SGSGX24YSX25IX26SGIEWAX27TNGMDVINMSLGGX28SGSX29AMKX30AVDNA YAX31GX32VVVAAAGNX33GX34SGNTNTX35GYPX36KYX37SVIAVGAVX38SNSX39X40ASFSSVGX41ELX42VMAPGX43X44X45X46 STYPX47NX48YX49X50X51NGTSMASPHVAGAAALILSKHPNLX52AX53QVX54X55X56LSX57TATYLGSX58FYYGKGLINVX59AAAQ. The letter X indicates that this site is a variable amino acid residue, containing any amino acid residue other than arginine, namely any one of the following: glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro), tryptophan (Trp), serine (Ser), tyrosine (Tyr), methionine (Met), cysteine ​​(Cys), phenylalanine (Phe), asparagine (Asn), glutamine (Gln), threonine (Thr), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), and histidine (His). The ArgR1 series protein backbone includes, but is not limited to, the variable sites X1 to X59. ArgR1 protein variants derived from this backbone are named ArgR1-1, ArgR1-2, etc. Taking into account both protein quality and expression levels, this embodiment only describes the two variants, ArgR1-1 and ArgR1-2 (with mature peptide sequences SEQ ID NO:1 and SEQ ID NO:2, respectively). The amino acid sequence identity between ArgR1-1 and ArgR1-2 is 86%.

[0034] 1. Amino acid composition analysis: In this embodiment, the amino acid content of ArgR1-1 and ArgR1-2 were analyzed and the essential amino acid scores were determined.

[0035] (1) Table 1 shows the content of essential and non-essential amino acids (g / 100 g protein) in ArgR1-1 and ArgR1-2, with the amino acid content in casein and whey protein, which are the most abundant in milk, plant-based soy protein, and human breast milk protein as references.

[0036] Table 1. Amino acid content analysis and comparison of proteins (g / 100 g protein)

[0037] (2) Essential amino acid score (AAS) To assess the nutritional value of the target protein, a score is awarded based on the content of essential amino acids in the protein sequence, namely histidine, threonine, valine, isoleucine, leucine, lysine, methionine, cysteine, phenylalanine, tyrosine, and tryptophan—a total of 11 amino acids. The calculation formula is: AAS = (AA content in the target protein (mg / g protein)) / (AA content in the ideal or reference protein (mg / g protein)) × 100. The ideal or reference protein score is derived from the 2013 report of the Food and Agriculture Organization of the United Nations (FAO), which provides baseline values ​​(mg / g protein) for essential amino acids for individuals aged 3 years and older.

[0038] Table 2 shows the AAS values ​​of essential amino acids for adults and infants in ArgR1-1 and ArgR1-2, with the AAS values ​​of casein and whey protein, which are the most abundant proteins in milk, plant-based soy protein, and human breast milk protein as references.

[0039] Table 2. Essential Amino Acid Score (AAS)

[0040] 2. Protease digestion analysis: In this embodiment, PeptideCutter (https: / / web.expasy.org / peptide_cutter / ) was used to perform online protease digestion analysis on the target proteins ArgR1-1 and ArgR1-2. The enzymes used included pepsin (under pH = 1.3 and pH > 2 conditions) and trypsin.

[0041] Table 3. Analysis of protease cleavage sites

[0042] Both ArgR1-1 and ArgR1-2 mature peptides consist of 274 amino acids. Predictive analysis indicates that after digestion with pepsin and trypsin, the degradation products of these two protein variants will contain 75% peptides and 81% residues with fewer than 5 amino acids. Online analysis with AlgPred showed no known IgE epitopes in the mature peptide sequences of ArgR1-1 and ArgR1-2.

[0043] 3. Protein expression and detection: This embodiment uses the Bacillus licheniformis expression system as an example. The recombinant Bacillus licheniformis strain CICC 10266 was used as the substrate strain. To improve the expression level and purity of the target protein, the CICC 10266 strain was modified by knocking out or silencing nine genes encoding extracellular secretory proteins, namely… cat, epr, bpr1, bpr2, apr, yhfN, SpoIIAC, lytC, mpr The resulting secretory protein-deficient strain was named 10266Δ9.

[0044] Based on the coding DNA sequences of protein variants ArgR1-1 (SEQ ID NO:1) and ArgR1-2 (SEQ ID NO:2) and the codon preference of the expression system, a gene was designed and synthesized in its entirety. ArgR1-1 and ArgR1-2 .

[0045] In this embodiment, the pHY300PLK plasmid was used as a vector, and a seamless cloning kit was used, following its operating procedures, to... ArgR1-1 and ArgR1-2 The recombinant plasmid pHY300PLK was assembled onto the pHY300PLK plasmid and, after screening and verification, was obtained. ArgR1-1 and pHY300PLK- ArgR1-2 .

[0046] The plasmid pHY300PLK- ArgR1-1 and pHY300PLK- ArgR1-2 In vitro methylation modification was performed. The main steps were: first, the plasmid was transformed into E. coli HST04 to complete demethylation, and then amplified to OD in LB medium containing ampicillin. 600 = 0.2~0.4, respectively, extract and purify the demethylated plasmid pHY300PLK- ArgR1-1 and pHY300PLK- ArgR1-2 In vitro methylation was performed in a buffer solution containing CpG methyltransferase and S-adenosylmethionine, incubated at 37°C for 1–2 h. After the reaction was complete, the plasmid was inactivated at 65°C, and the methylated plasmid pHY300PLK- was recovered and purified. ArgR1-1 and pHY300PLK- ArgR1-2 .

[0047] Take an appropriate amount of the methylated plasmid pHY300PLK- ArgR1-1 and HY300PLK- ArgR1-2 The recombinant bacterial strain 10266Δ9 was electroporated. The electroporation conditions were: 1 mm electroporation cuvette, 2.1 KV once, time 4.5–5.0 ms. The electroporated strains were named 10266Δ9-ArgR1-1 and 10266Δ9-ArgR1-2. A suitable amount of bacterial culture was streaked onto LB agar plates containing 10 μg / mL tetracycline to isolate single colonies, which were then transferred to 250–500 mL of liquid LB medium (containing 10 μg / mL tetracycline) and incubated in shake flasks at 37°C for 24–72 hours. The fermentation broth was collected, and the target protein concentration (g / L fermentation broth) was determined using the Bradford assay combined with SDS-PAGE.

[0048] Table 4. Protein expression detection

[0049] Example 2: Design and expression of Arginine-free ArgR2 series proteins This example uses *Trichoderma reesei* to express arginine-free ArgR2 series recombinant proteins. These ArgR2 series protein variants share the same protein backbone, and their mature peptide sequences are as follows: QTMCSQYX1SASSPPYSVNQNLWGX2YQGTGSQCVYVX3KLSSSGASWHTKWTWSGGX4GTVKSYSNSGLTFDKKLX5SDVSSX6PTSVKWSQDDTNVQADVSYDLFTAANADHATSSGDY ELMIWLAX7YGTVQPIGKQIATATVGGKSWEVWYGTSX8QAGAEQKTYSFVAGSPX9NSYSGDX10KX11FFNYLTQNQGFPASSQHLITLQFGTX12PFTGGPATFTX13X14NWTASVN.

[0050] The letter X indicates that the site is a variable amino acid residue, containing amino acid residues other than arginine (same explanation as in Example 1). The ArgR2 series protein backbone includes, but is not limited to, the variable sites X1~X14. ArgR2 protein variants derived from this backbone are named ArgR2-1, ArgR2-2, etc. Since most protein variants have very low expression levels in *Trichoderma reesei*, considering both protein quality and expression level, this example only describes the two variants, ArgR2-1 and ArgR2-2 (mature peptide sequences SEQ ID NO:3 and SEQ ID NO:4, respectively). The amino acid sequence identity between ArgR2-1 and ArgR2-2 is 94.2%.

[0051] 1. Amino acid composition analysis: In this embodiment, the amino acid content of ArgR2-1 and ArgR2-2 were analyzed and the essential amino acid score was determined, using the same method as in Example 1.

[0052] (1) Analysis of amino acid content.

[0053] Table 5. Amino acid content analysis and comparison of proteins (g / 100 g protein)

[0054] (2) Essential amino acid score (AAS) Table 6. Essential Amino Acid Score (AAS)

[0055] 2. Protease digestion analysis: Online protease digestion analysis was performed on the target proteins ArgR2-1 and ArgR2-2, following the method described in Example 1.

[0056] Table 7. Analysis of protease cleavage sites

[0057] Both ArgR2-1 and ArgR2-2 mature peptides consist of 223 amino acids. Predictive analysis indicates that after digestion with pepsin and trypsin, the degradation products of these two protein variants will contain 85% peptides and 81% residues with 5 or fewer amino acids, respectively. Online analysis with AlgPred showed no known IgE epitopes in the mature peptide sequences of ArgR2-1 and ArgR2-2.

[0058] 3. Protein expression and detection: This embodiment uses the Trichoderma reesei expression system as an example.

[0059] (1) Expression plasmid construction: Based on the coding DNA sequences of protein variants ArgR2-1 (SEQ ID NO:3) and ArgR2-2 (SEQ ID NO:4) and the codon preference of the expression system, the expression plasmid was designed and synthesized in its entirety. ArgR2-1 and ArgR2- 2 Using pMDT05-cbh1-KO plasmid as a vector, and following the instructions of the seamless cloning kit, the plasmid was cloned... ArgR2-1 and ArgR2-2 The recombinant plasmid pMDT05-cbh1-KO was assembled onto the pMDT05-cbh1-KO plasmid and, after screening and verification, was obtained. ArgR2-1 and pMDT05- ArgR2-2 .

[0060] (2) Gene integration and screening of high-expression strains: The above recombinant plasmid pMDT05- ArgR2-1 and pMDT05- ArgR2-2 Agrobacterium was transformed separately, and the resulting Agrobacterium was then transformed into the Trichoderma reesei auxotrophic strain Rut-C30. pyr4 (cbh1). After transforming Agrobacterium, a small number of hyphae were picked and identified by PCR after culturing on PDA plates for 3-4 days. Successfully integrated strains were prepared into spore suspensions using sterile water and cultured on PDA plates containing 0.1% Triton X-100 at 28°C for 2-3 days until single colonies could be isolated. Single colonies were transferred to PDA medium and cultured at 28°C for 3-4 days. Hyphae were then picked and identified by PCR. Positive strains were cultured until spore maturation on day 7.

[0061] Mature spores were collected and cultured on PDA plates containing 10 mM uridine, 5 mg / mL 5-FOA, and 0.1% Triton X-100 at 28°C for 4-6 days. Single colonies were picked and transferred to PDA plates containing 10 mM uridine for sporulation culture at 28°C. After 3-4 days of culture, hyphae were picked for PCR verification. Successfully integrated cells were selected, and spore suspensions were prepared using sterile water. These suspensions were cultured on PDA plates containing 0.1% Triton X-100 and 10 mM uridine at 28°C for 2-3 days. Single colonies were transferred to PDA medium containing 10 mM uridine and cultured at 28°C for 3-4 days. Hyphae were picked for PCR verification. Positive strains were cultured until spore maturation on day 7. Positive strains were named Rut-C30. / ArgR2-1 and Rut-C30 / ArgR2-2.

[0062] (3) Protein expression detection: The culture medium consisted of 7.5 g / L glucose, 37 g / L whey powder, 40 g / L soybean meal, 21.9 g / L potassium dihydrogen phosphate, 7.5 g / L ammonium sulfate, 1.5 g / L magnesium sulfate heptahydrate, 0.5 g / L calcium chloride, and 0.2 mL / L inorganic salt nutrient solution. The initial pH was 4.5-5.0, and the temperature was 28-30°C. After culturing for 120 h, the fermentation supernatant was collected, and the target protein was purified using an ion chromatography column (DEAE-Sepharose FF). The concentration of the purified protein (g / L fermentation broth) was determined by Bradford assay combined with SDS-PAGE.

[0063] Table 8. Protein expression detection

[0064] Example 3: Design and expression of Arginine-free ArgR3 series proteins This example uses Pichia pastoris to express arginine-free ArgR3 series recombinant proteins. These ArgR3 series protein variants have the same protein backbone, and their mature peptide sequences are as follows: The letter X indicates that the site is a variable amino acid residue, containing amino acid residues other than arginine (same explanation as in Example 1). The ArgR3 series protein backbone includes, but is not limited to, the variable sites X1 to X20. ArgR3 protein variants derived from this backbone are named ArgR3-1, ArgR3-2, etc. Since most protein variants are expressed at very low levels in Pichia pastoris, considering both protein quality and expression level, this example only describes the two variants, ArgR3-1 and ArgR3-2 (mature peptide sequences SEQ ID NO:5 and SEQ ID NO:6, respectively). The amino acid sequence identity between ArgR3-1 and ArgR3-2 is 95.9%.

[0065] 2. Amino acid composition analysis: In this embodiment, the amino acid content of ArgR3-1 and ArgR3-2 were analyzed and the essential amino acid score was determined, using the same method as in Example 1.

[0066] (1) Analysis of amino acid content.

[0067] Table 9. Analysis and comparison of amino acid content of proteins (g / 100 g protein)

[0068] (2) Essential amino acid score (AAS) Table 10. Essential Amino Acid Score (AAS)

[0069] 2. Protease digestion analysis: Online protease digestion analysis was performed on the target proteins ArgR3-1 and ArgR3-2, and the specific method is described in Example 1.

[0070] Table 11. Analysis of protease cleavage sites

[0071] Both ArgR3-1 and ArgR3-2 mature peptides consist of 508 amino acids. Predictive analysis indicates that after digestion with pepsin and trypsin, peptides and residues of 5 or fewer amino acids constitute 86% of the degradation products for both protein variants. Online analysis with AlgPred revealed no known IgE epitopes in the mature peptide sequences of ArgR3-1 and ArgR3-2.

[0072] 3. Protein expression and detection: This embodiment uses the Pichia pastoris expression system as an example.

[0073] (1) Expression plasmid construction: Based on the coding DNA sequences of protein variants ArgR3-1 (SEQ ID NO:5) and ArgR3-2 (SEQ ID NO:6) and the codon preference of the expression system, the expression plasmid was designed and synthesized in its entirety. ArgR3-1 and ArgR3- 2 In this embodiment, pGAPZαA plasmid was used as a vector, and a seamless cloning kit was used, following its operating procedures, to... ArgR3-1 and ArgR3-2 The recombinant plasmid pGAPZαA was assembled onto the pGAPZαA plasmid and, after screening and verification, was obtained. ArgR3-1 and pGAPZαA- ArgR3-2 After identification and purification, store at -80°C for later use.

[0074] (2) Preparation of competent cells of Pichia pastoris X33: After activating Pichia pastoris X33, an appropriate amount of bacterial culture was inoculated into YPD medium and cultured overnight. The cells were collected in pre-chilled centrifuge tubes, centrifuged at 4℃ / 1500 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in a buffer solution containing 100 mM LiAc, 10 mM DTT, 600 mM sorbitol, and 10 mM pH 7.5 Tris-HCl, placed at room temperature for 30 min, and centrifuged at 4℃ to collect the cells. The cells were resuspended in pre-chilled sorbitol solution (concentration of 1 M), washed twice at 4℃, and then resuspended in pre-chilled sorbitol solution (concentration of 1 M) and dispensed to achieve a cell concentration of ~10. 10 per mL.

[0075] (3) Obtaining recombinant strains: Take the above plasmid pGAPZαA- ArgR3-1 and pGAPZαA- ArgR3-2 The X33 competent cells were linearized using the restriction endonuclease Avr II (Bln I), purified, and then electroporated. The electroporation conditions were: 2 mm electroporation cuvette, 1.5 KV once, time 4.5–5.0 ms. Immediately after electroporation, 1 M sorbitol was added, and the cells were incubated at 30°C for 1 h. After centrifugation at 4°C, the supernatant was discarded, and the cells were resuspended. A suitable amount of the bacterial suspension was plated onto YPD plates containing 100 μg / mL bleomycin. The cells were incubated at 30°C until visible colonies appeared. Positive strains were identified by colony PCR and named X33-ArgR3-1 and X33-ArgR3-2. The positive colonies were inoculated into YPD medium containing 100 μg / mL bleomycin and cultured in shake flasks at 30°C for 20 h. After centrifugation, the supernatant was discarded, 30% glycerol was added, and the cells were stored at -80°C.

[0076] (4) Protein expression detection: After activating the above-mentioned positive bacteria X33-ArgR3-1 and X33-ArgR3-2, they were inoculated into YPD plates containing 100 μg / mL bleomycin and streaked. Single colonies were picked and cultured overnight at 30℃ in 10 mL of YPD. Then, they were inoculated at a ratio of 10% into optimized YPD medium (1.32% yeast extract, 3.48% glucose, 2% peptone, pH 6.86) and cultured at 25.6℃ in shake flasks for 2-3 days. The fermentation broth was collected by centrifugation at 4℃. 250 mL of fermentation broth was heated to 75℃, centrifuged to remove heat-sensitive proteins, and the supernatant was recovered. The concentration of the target protein (g / L fermentation broth) was detected by Bradford assay combined with SDS-PAGE.

[0077] Protein purification and detection: After collecting the fermentation supernatant, the pH was adjusted to 2.5-3.0, centrifuged to remove most of the acid-sensitive impurity proteins, and the supernatant was collected. The concentration of the target protein (g / L fermentation broth) was determined by Bradford assay combined with SDS-PAGE.

[0078] Table 12. Protein expression detection

[0079] Example 4: Design and expression of Arginine-free ArgR4 series proteins This example uses Bacillus subtilis to express arginine-free ArgR4 series recombinant proteins. These ArgR4 series protein variants share the same protein backbone, and their mature peptide sequences are as follows: MAFSX1X2SAQAAGKSX3X4EKKYIX5GFKQTMSX6X7SSAKKKDX8ISEKGGKVQKQFKYVX9AAX10ATLX11EKAVKELKKDPX12VAYVEEDHX13AHX14YAQSVPYGX15 SQIKAPALHSQGYTGSNVKVAX16IDSGIDSSHPDLX17VX18GGASX19VPSETNPX20QDGX21SHGTHVAGTX22AALNNSIGVLGVAPSASLYAVKVLX23X24X25GSGQY SWIINGIEWAIX26NNMDVINMSLGGPX27GSX28ALKX29X30VDKAVX31SGX32VVX33AAAGNEGX34SGSX35STVGYPX36KYPSX37IAVGAVX38SSNQX39ASFSSX40GX41ELDVMAPGVSIQSTLPGX42X43YGAYNGTSMAX44PHX45AGAAALILSKHPX46WTNX47QVX48X49X50LEX51TX52TX53LGX54SFYYGKGLINX55QAAAQ. The letter X indicates that the site is a variable amino acid residue, containing amino acid residues other than arginine (same explanation as in Example 1). The ArgR4 series protein backbone includes, but is not limited to, variable sites X1~X55. ArgR4 protein variants derived from this backbone are named ArgR4-1, ArgR4-2, etc. Since most protein variants are expressed at very low levels in Bacillus subtilis, considering both protein quality and expression levels, this embodiment only describes the two variants, ArgR4-1 and ArgR4-2 (mature peptide sequences SEQ ID NO:7 and SEQ ID NO:8, respectively). The amino acid sequence identity between ArgR4-1 and ArgR4-2 is 84.5%.

[0080] 3. Amino acid composition analysis: In this embodiment, the amino acid content of ArgR4-1 and ArgR4-2 were analyzed and the essential amino acid scores were determined, using the same methods as in Example 1.

[0081] (1) Analysis of amino acid content.

[0082] Table 13. Amino acid content analysis and comparison of proteins (g / 100 g protein)

[0083] (2) Essential amino acid score (AAS) Table 14. Essential Amino Acid Score (AAS)

[0084] 2. Protease digestion analysis: Online protease digestion analysis was performed on the target proteins ArgR4-1 and ArgR4-2, following the method described in Example 1.

[0085] Table 15. Analysis of protease cleavage sites

[0086] Both ArgR4-1 and ArgR4-2 mature peptides consist of 362 amino acids. Predictive analysis indicates that after digestion with pepsin and trypsin, peptides and residues of 5 or fewer amino acids will account for 73% of the degradation products of both protein variants. Online analysis with AlgPred shows that the mature peptide sequences of ArgR4-1 and ArgR4-2 contain no known IgE epitopes.

[0087] 3. Protein expression and detection: This embodiment uses the Bacillus subtilis expression system as an example, employing the secretory Bacillus subtilis strain WB800N (nprE aprE epr bpr mpr :: ble nprB :: bsr .vpr wprA :: hyg cm :: neo; NeoR). Based on the coding genes and expression system preferences, the expression gene was designed and synthesized in its entirety. ArgR4-1 and ArgR4-2 The plasmids were assembled into the *E. coli*-*Bacillus subtilis* shuttle plasmid PHT01 using a seamless cloning method. After transformation into *E. coli* DH5α competent cells and screening, positive recombinant plasmid PHT01- was obtained. ArgR4-1 and PHT01- ArgR4-2 After purification, the plasmid is stored at -20℃ for later use.

[0088] Prepare Bacillus subtilis electrotransformation competent cells, referring to the product manual for specific procedures. After aliquoting, store at -80℃ for later use. Take the above recombinant plasmid PHT01- ArgR4-1and PHT01- ArgR4-2 The competent cells WB800N were electroporated. The electroporation conditions were: 1 mm electroporator, 2.0 KV once, time 4.5–5.0 ms. Immediately add 1 mL of LB resuscitation medium containing 0.5 M sorbitol and 0.38 M mannitol, and incubate at 37°C with shaking for 3–4 h. After resuscitation, centrifuge and resuspend the cells, then plate them onto LB agar plates (containing 10 μg / mL chloramphenicol) and incubate at 37°C for 1–2 days. Positive strains were screened by colony PCR and named WB800N- ArgR4-1 and WB800N- ArgR4-2 Store frozen at -80℃ for later use.

[0089] Activation of the above recombinant Bacillus subtilis WB800N- ArgR4-1 and WB800N- ArgR4-2 After subculturing, seed culture was inoculated at 1% into LB (containing 20 μg / mL chloramphenicol) and cultured at 37°C until OD600 = 0.6–0.8. Then, 1 mM IPTG was added for induction culture for 2–4 h. The bacterial culture was centrifuged at 4 °C for 10 min, and the supernatant crude protein solution was collected. After filtration through a 0.22 μm filter membrane, the protein was analyzed using SDS-PAGE and a Bradford protein quantification kit.

[0090] Table 16. Protein Expression Detection

[0091] Example 5: Design and expression of Arginine-free ArgR5 series proteins This example uses Pichia pastoris to express arginine-free ArgR5 series recombinant proteins. These ArgR5 series protein variants have the same protein backbone, and their mature peptide sequences are as follows: MGAFTEKQEALVNSSWEAFKGNIPQYSVVFYTSILEKAPAAKNLFSFLANGVDPTNPKLTX1HAEX2LFGLVX3DSAX4QLX5ANGX6VVADAALGSIHX7QKX8X9X10DX11QFX12VVKEALLKTX13KEAVGDKWX14DELSX15AX16EX17AYDELAAAIKKAFX18. The letter X represents a variable amino acid residue at that site, containing amino acid residues other than arginine (same explanation as in Example 1). The ArgR5 series protein backbone includes, but is not limited to, variable sites X1~X18. ArgR5 protein variants derived from this backbone are named ArgR5-1, ArgR5-2, etc. Since most protein variants are expressed at very low levels in Bacillus subtilis, this embodiment only uses the two variants, ArgR5-1 and ArgR5-2 (with mature peptide sequences SEQ ID NO:9 and SEQ ID NO:10, respectively), as examples, taking into account both protein quality and expression level. The amino acid sequence identity between ArgR5-1 and ArgR5-2 is 87.7%.

[0092] 4. Amino acid composition analysis: In this embodiment, the amino acid content of ArgR5-1 and ArgR5-2 were analyzed and the essential amino acid score was determined, using the same method as in Example 1.

[0093] (1) Analysis of amino acid content.

[0094] Table 17. Amino acid content analysis and comparison of proteins (g / 100 g protein)

[0095] (2) Essential amino acid score (AAS) Table 18. Essential Amino Acid Score (AAS)

[0096] 2. Protease digestion analysis: Online protease digestion analysis was performed on the target proteins ArgR5-1 and ArgR5-2, and the specific method is described in Example 1.

[0097] Table 19. Analysis of protease cleavage sites

[0098] The mature peptides of ArgR5-1 and ArgR5-2 consist of 146 and 145 amino acids, respectively. Predictive analysis indicates that after digestion with pepsin and trypsin, the degradation products of these two protein variants will contain peptides and residues with 5 or fewer amino acids, accounting for 89% and 84%, respectively. AlgPred prediction analysis shows that the mature peptide sequences of ArgR5-1 and ArgR5-2 contain no known IgE epitopes.

[0099] 3. Protein expression and detection: This embodiment uses the Pichia pastoris expression system as an example. The methods for constructing expression plasmids, gene integration, screening for high-expression positive strains, shaking-flask culture of recombinant strains, and expression of the target proteins ArgR5-1 and ArgR5-2 are as described in Example 3.

[0100] Protein purification and detection: After collecting the fermentation supernatant, the target protein was purified by anion exchange chromatography. A DEAE cellulose anion exchange column was used for gradient elution with NaOAc buffer at pH 5.4. The HPLC UV monitor was set to 280 / 405 nm, and the target protein was collected. The expression level of the target protein was determined by SDS-PAGE and Coomassie Brilliant Blue colorimetry.

[0101] Table 20. Protein Expression Detection

[0102] Example 6: Design and expression of Arginine-free ArgR6 series proteins This example uses *E. coli* to express arginine-free ArgR6 series recombinant proteins. These ArgR6 series protein variants have the same protein backbone, and their mature peptide sequences are as follows: KQFTKCEX1SQX2LKDX3DGYGGX4AX5PELX6CTMFHTSGYDTQAIVQNNDSTEYGLFQIX7NKX8WCKX9X10QX11PX12SX13NICX14ISCDKFLDDDX15TDDIMCX16KKILDX17X18GX19X20YWLAHKALCX21EKLX22QWLCEKL. The letter X represents a variable amino acid residue at that site, including amino acid residues other than arginine (same explanation as in Example 1). The ArgR6 series protein backbone includes, but is not limited to, variable sites X1~X22. ArgR6 protein variants derived from this backbone are named ArgR6-1, ArgR6-2, etc. Since most protein variants are expressed at very low levels in Bacillus subtilis, this embodiment only describes the two variants, ArgR6-1 and ArgR6-2 (with mature peptide sequences SEQ ID NO:11 and SEQ ID NO:12, respectively), taking into account both protein quality and expression level. The amino acid sequence identity between ArgR6-1 and ArgR6-2 is 78.9%.

[0103] 5. Amino acid composition analysis: In this embodiment, the amino acid content of ArgR6-1 and ArgR6-2 were analyzed and the essential amino acid score was determined, using the same method as in Example 1.

[0104] (1) Analysis of amino acid content.

[0105] Table 21. Amino acid content analysis and comparison of proteins (g / 100 g protein)

[0106] (2) Essential amino acid score (AAS) Table 22. Essential Amino Acid Score (AAS)

[0107] 2. Protease digestion analysis: Online protease digestion analysis was performed on the target proteins ArgR6-1 and ArgR6-2, and the specific method is described in Example 1.

[0108] Table 23. Analysis of protease cleavage sites

[0109] The mature peptides of ArgR6-1 and ArgR6-2 consist of 123 amino acids. Predictive analysis indicates that after digestion with pepsin and trypsin, the degradation products of these two protein variants will contain peptides and residues with 5 or fewer amino acids, accounting for 87% and 82%, respectively. AlgPred prediction analysis shows that the mature peptide sequences of ArgR6-1 and ArgR6-2 contain no known IgE epitopes.

[0110] 3. Protein expression and detection: The example uses the Escherichia coli expression system, and the Escherichia coli K12 used was purchased from the American Type Culture Collection (ATCC).

[0111] (1) Construction of expression carrier: Based on coding genes and expression system preferences, design and synthesize whole-genome expression genes. ArgR5-1 and ArgR5-2 The plasmids were assembled into the pET22b plasmid using a seamless cloning method. The plasmids were then transformed into *E. coli* DH5α competent cells. Single colonies were picked from LB agar plates (containing 100 μg / mL ampicillin) for screening by colony PCR. Positive strains identified by sequencing were used to extract the plasmid, which was named pET- ArgR5-1 and pET- ArgR5-2 After purification, store at -20℃ for later use.

[0112] (2) Transformation of recombinant plasmids: The pET- ArgR5-1 and pET- ArgR5-2 The plasmids were transformed into E. coli K12 competent cells, cultured and screened on LB agar plates containing 100 μg / mL ampicillin, and identified by PCR and sequencing to determine positive expression strains, which were named K12- ArgR5-1 and K12- ArgR5-2 Store frozen at -80℃ for later use.

[0113] Activate the K12- ArgR5-1 and K12- ArgR5-2 For the frozen bacteria, streak LB agar plates (containing 100 μg / mL ampicillin), pick single colonies, and inoculate them into 50 mL LB medium (100 μg / mL ampicillin). Incubate overnight at 37°C with shaking until OD600 reaches 1%. Alternatively, inoculate 1% of the culture into LB liquid medium (100 μg / mL ampicillin), incubate at 22°C with shaking until OD600 = 0.6–0.8, add 0.5 mM IPTG, and induce incubation overnight at 22°C.

[0114] (3) Recombinant protein expression and detection: After fermentation (12-16 h), the supernatant was collected by centrifugation at 4°C, and then incubated in an 85°C water bath for 30-60 min to remove heat-sensitive proteins. The supernatant was collected by centrifugation at 12,000 rpm for 20-30 min at 4°C. The concentration of the target protein (g / L fermentation broth) was determined by Bradford assay combined with SDS-PAGE.

[0115] Table 24. Protein Expression Detection

[0116] Example 7: Preparation of protein powder with / without arginine 1. Gene integration: (1) Bacillus expression system: This section describes the process of integrating recombinant protein-coding genes into the genome of a Bacillus expression system, referring to Examples 1 and 4. This example uses ArgR1-1 as an example; other sequences can be operated on using the same method as described below: Based on the coding gene sequence and the codon preference of expression system strain 10266Δ9, a whole-genome gene was designed and synthesized. Using a seamless cloning kit, following the manufacturer's instructions, the synthesized target genome was inserted into the catUD-pEBKan194-GFP plasmid, with the target gene positioned cis-registered between the upstream and downstream homologous arms of cat (i.e., catU and catD sequences). The cells were transformed into *E. coli* DH5α competent cells, selected with 20 μg / mL kanamycin, and single colonies were picked for colony PCR and sequencing identification. Plasmids were extracted from colonies with correct sequencing results and named pEBKan194-. ArgR1 Store at -20℃ for later use.

[0117] Next, the construction and screening of gene-integrated recombinant strains were carried out. The plasmid pEBKan194- ArgR1 Transform strain 10266Δ9 and use the homologous double crossover method to transfer the target gene. ArgR1-1 The original strain integrated into the strain cat Gene locus. After sequencing and identification, it was integrated with... ArgR1-1 The recombinant strain containing the gene was cryopreserved and named ArgR1-1(cat_i). Using the same method, the target gene was integrated into the remaining eight sites in strain 10266Δ9 where the original gene had been knocked out, resulting in eight recombinant strains.

[0118] The obtained recombinant strains were inoculated into 25–50 mL of LB medium and cultured at 37°C with shaking at 220 rpm for 24–72 hours. After purification, the expression level of the recombinant protein was detected. Two to five integration sites with the highest expression levels were selected, and recombinant strains with two, three, or higher copy numbers were constructed according to the above method.

[0119] (2) Trichoderma reesei expression system: This section relates to Example 2, in which the gene encoding the protein variant has been integrated into the genome of Trichoderma reesei, as detailed in Example 2.

[0120] (3) Yeast expression system This section relates to Examples 3 and 5, where the coding gene for the protein variant has been integrated into the genome of Pichia pastoris, as detailed in the respective examples.

[0121] (4) Escherichia coli expression system: This section relates to Example 6, in which the protein variant is expressed in E. coli via a free plasmid and may or may not be integrated into the host genome.

[0122] 2. Medium-scale fermentation expression and preparation using recombinant strains: (1) Bacillus expression system: The fermentation process of recombinant strains using Bacillus licheniformis, Bacillus subtilis, and other Bacillus species as expression systems is as follows, specifically involving Examples 1 and 4: Fermentation was carried out in a 7-liter fermenter using a continuous fed-batch process. The basic fermentation medium contained: 3.0% corn starch, 2.5% soy protein isolate, 0.25% yeast extract, 40.45% dipotassium hydrogen phosphate, 0.9% sodium dihydrogen phosphate, 0.04% calcium chloride, 0.08% magnesium sulfate heptahydrate, 0.02% manganese sulfate, 0.2% antifoaming agent, and 0.01% α-thermo-amylase. The pH was adjusted to 7.5, and the medium was sterilized at 121°C for 20 minutes. The fed-batch medium contained: 40% glucose, 18% hydrolyzed soy protein, and 2% yeast extract. Both were sterilized at 121°C. The inoculum size was 5%, and the optimized culture conditions were 37℃, aeration rate of 0.6~1.2 vvm, and dissolved oxygen level was controlled above 40% by adjusting the stirring speed and aeration rate. Feeding began after 12 h of fermentation, with the following feeding rates: glucose 1.8~2.5 g / L / h, soybean hydrolyzed protein + yeast powder 0.7~0.9 g / L / h, ammonia water to maintain pH 6.75~6.80, and a feed rate of 0.7~1.0 mL / L / h. Fermentation ended after 72 h, with a total protein concentration of 10.5~13.5 g / L.

[0123] (2) Filamentous fungal expression system: The fermentation process of the recombinant strain using Trichoderma as the expression system is as follows, specifically involving Example 2: The recombinant strain was inoculated into PDA plates and cultured at 28°C for 7 days. A spore suspension (~1.0 × 10⁻⁶) was then collected and prepared. 8 The seed culture (1% inoculum per mL) was inoculated into seed culture medium at 28°C / 170 rpm in the dark with shaking for 24–36 h. The seed culture was then transferred to basal fermentation medium at a ratio of 10%, in 28 L / 50 L fermenters. The basal culture medium consisted of: glucose 20 g / L, corn steep liquor powder 7 g / L, urea 1 g / L, potassium dihydrogen phosphate 4 g / L, ammonium sulfate 2 g / L, calcium chloride 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, manganese chloride 1 mM, trace elements 1 mL / L, and pH 4.0. The medium was aerated and stirred at 28°C, with the rotation speed controlled to maintain dissolved oxygen above 30% and pH 3.5–4.0. After 24–28 h of culture, the carbon source was largely depleted, and 250 g / L lactose solution was added. After approximately 72 h of culture, the culture entered the induction expression stage. Add 250 g / L lactose solution, aerate and stir at 28°C, controlling the stirring speed to ensure the working concentration does not exceed 2 g / L and the dissolved oxygen level is not lower than 20%. Incubate for approximately 188 hours post-inoculation, taking samples every 24 hours to determine protein concentration. The total protein concentration of the fermentation broth should be 8.5–12.5 g / L.

[0124] (3) Yeast expression system: The fermentation process of the recombinant strain using yeast as the expression system is as follows, specifically involving Examples 3 and 5: First, the recombinant strain was inoculated into BMGY medium at a loading volume of 200 mL / 2L baffled shake flask. It was cultured at 28℃ / 220 rpm with shaking until OD600 = 4~6, then transferred at a 10% ratio to FM22 basal salt medium, with an initial loading volume of 2L / 5L fermenter. The cell growth stage temperature was 30℃, the induction stage temperature was 28℃, the stirring speed was 500~800 rpm, the dissolved oxygen content was >20%, and the pH was controlled to ~6.0 by adding ammonia and phosphoric acid. Fed-batch feeding was performed. When glycerol was depleted, 250~500 mL of 50% glycerol was added. To control dissolved oxygen, anhydrous methanol was added to a final concentration of 0.5% (V / V), and the methanol flow rate was adjusted according to dissolved oxygen fluctuations. Samples were taken every 6-8 h to detect cell wet weight and protein concentration. When fermented for 200 h, the total extracellular protein concentration was 5.5~9.6 g / L.

[0125] (4) Escherichia coli expression system: The fermentation process of the recombinant strain using *Escherichia coli* as the expression system is as follows, specifically referring to Example 6: After activation, the recombinant strain was cultured on LB agar plates containing 100 μg / mL ampicillin, and single colonies were isolated. These colonies were then inoculated into LB primary seed medium and cultured at 37°C / 220 rpm with shaking for 3-6 h. The culture was then transferred at a ratio of 3-5% to 2L LB secondary seed medium and cultured at 37°C / 220 rpm with shaking for 14-16 h. Finally, the culture was inoculated into a 30 L fermenter at a ratio of 3-5%. The optimized fermentation medium formulation included: glucose 10-20 g / L, yeast extract 10-15 g / L, peptone 5-10 g / L, sodium chloride 5-10 g / L, potassium dihydrogen phosphate 1-5 g / L, ammonium sulfate 1-5 g / L, magnesium sulfate 1-5 g / L, glycerol 5-10 g / L, and DF104 antifoaming agent 1-5 g / L, adjusting the pH to 7.5. The optimized fed-batch culture medium consisted of 100–500 g / L glucose and 100–200 g / L yeast extract. Fermentation was carried out until the OD600 reached approximately 10, then 0.5 mM IPTG was added for induction. Fermentation was induced at 37°C and pH 6.5–7.0 for 6–8 h, followed by fed-batch culture with dissolved oxygen maintained at 30–70%. After 20–36 h of fermentation, protein yield reached 3.6–5.5 g / L.

[0126] 4. Protein purification, drying, and preparation of protein powder: (1) The protein purification and drying process after fermentation using recombinant strains with Bacillus licheniformis, Bacillus subtilis, and other Bacillus species as expression systems is as follows, specifically involving Examples 1 and 4: The fermentation broth was separated into solid and liquid phases using a ceramic membrane to obtain the fermentation filtrate. 1% sodium chloride was added, and the pH was adjusted to 1.5-1.8 using hydrochloric acid. Protein was precipitated for 15-18 hours. After removing the supernatant, the concentrated protein precipitate was collected and washed with a ceramic membrane until the pH reached 2.7-2.9. The concentrated filtrate was collected, and the pH was adjusted to 9.0. After another 8-10 times volume of ceramic membrane treatment, the concentrated filtrate was collected, sterilized by pasteurization, and spray-dried to obtain protein powder.

[0127] Spray drying: The protein solution with a solid content of 1.3% is pasteurized at 60°C and sprayed into powder using a centrifugal spray dryer with an atomizing disc speed of 24,000 to 40,000 r / min, an inlet air temperature of 160°C, and an outlet air temperature of 75°C.

[0128] The obtained protein spray-dried powder was subjected to protein content determination by the Kjeldahl method (Method I) (GB 5009.5-2010 "Determination of Protein in Food"). The purity of the target protein was detected by SDS-PAGE and reversed-phase high-performance liquid chromatography. The amino acid content was detected according to the method of GB / T5009.124-2016 "Determination of Amino Acids in Food".

[0129] Testing revealed that the obtained protein powder had a total protein content of 85.5%–91.5%, a protein purity of 80.0%–88.6%, a moisture content of 5.03%–6.14%, and an arginine content of 2.5–7.5 mg / g protein powder. Further optimization of the technical process in this embodiment could improve both protein purity and yield.

[0130] (2) The protein purified and dried after fermentation by the recombinant strain using filamentous fungi as the expression system is as follows, specifically referring to Example 2: After fermentation in the tank, 1% perlite was added to the broth, and the mycelium was removed by plate and frame filtration. The filtrate was collected. 0.5% activated carbon was added to the filtrate, stirred evenly, and allowed to stand for 10-15 minutes for decolorization. Then, 1% perlite was added, mixed well, and the mixture was filtered again to remove the decolorizing activated carbon. The filtrate was collected. The filtrate was filtered through a 0.45 μm pleated filter cartridge, and the permeate was collected. The clarified permeate was adjusted to pH 2.0-2.5 by adding phosphoric acid while stirring. The mixture was allowed to stand at room temperature for 3 hours to precipitate the protein, and the supernatant was discarded. The target protein was precipitated and concentrated, washed three times with pure water through a 200 nm inorganic ceramic membrane, and then concentrated to 30-50 g / L. The concentrated protein was spray-dried (under the same conditions as above), and the resulting protein powder was used for protein content determination (using the same method as above).

[0131] Testing revealed that the obtained protein powder had a total protein content of 78.6-85.5%, a protein purity of 77.5-84.5%, a moisture content of 4.32-5.30%, and an arginine content of 3.1-8.6 mg / g protein powder. Further optimization of the technical process in this embodiment could improve both protein purity and yield.

[0132] (3) The protein purified and dried after fermentation by the recombinant strain using yeast as the expression system is as follows, specifically referring to Examples 3 and 5: The fermentation broth was centrifuged using a disc centrifuge to separate the solid and liquid components. The resulting supernatant was treated with a 200 nm ceramic membrane to obtain the fermentation filtrate. The pH was adjusted to 1.3–2.0 with HCl, and the protein was allowed to settle for 15–18 hours. After removing the supernatant, the concentrated protein precipitate was collected. The precipitate was then washed with a ceramic membrane at a volume of 54 times, and the concentrated protein was collected. The concentrated protein was spray-dried (under the same conditions as above), and the resulting protein powder was used for protein content determination (using the same method as above).

[0133] Testing revealed that the obtained protein powder had a total protein content of 83.5-90.3%, a protein purity of 76.2-83.5%, a moisture content of 5.65-6.05%, and an arginine content of 4.2-9.8 mg / g protein powder. Further optimization of the technical process in this embodiment could improve both protein purity and yield.

[0134] (4) The protein purification and drying process after fermentation using Escherichia coli as the expression system is as follows, specifically referring to Example 6: The fermentation broth was collected and centrifuged, the supernatant was discarded, and the bacterial sludge was washed with 10 mM borate buffer (pH 9.0). The sludge was then lysed using a high-pressure homogenizer, centrifuged at 10,000 g, and the supernatant was collected. The pH was adjusted to 5.5, and the precipitate was collected by centrifugation. The precipitate was washed with borate buffer, and the pH was adjusted to 5.5 to precipitate the protein. After washing 3-5 times, the concentrated protein was collected. The concentrated protein was spray-dried (under the same conditions as above), and the resulting protein powder was used for protein content determination (using the same method as above).

[0135] Testing revealed that the obtained protein powder had a total protein content of 78.6-85.0%, a protein purity of 72.5-80.0%, a moisture content of 6.04-6.78%, and an arginine content of 6.3-10.0 mg / g protein powder. Further optimization of the technical process in this embodiment could improve both protein purity and yield.

[0136] Example 8: Feeding rats with arginine-free / low-arginine protein powder can reduce the arginine content in rat plasma. In this embodiment, the experimental animals were male Sprague-Dawley rats aged 8-10 weeks, weighing 250 ± 25 g, housed in an SPF-grade animal facility, and kept in strict accordance with the requirements of the Chinese Guidelines for Ethical Review of Laboratory Animal Welfare (GB / T35892-2018) during the experiment. The rats had free access to feed and distilled water during the experiment.

[0137] After acclimatizing to their environment for one week with normal adult rat diet and distilled water, the experimental animals were randomly divided into groups of 3-5 rats each. The experimental design was as follows: blood was collected from the tail vein of the stabilized rats to obtain plasma for baseline data acquisition. Control rat diet and arginine-restricted rat diet were distributed to each group of rats. Blood was collected from the tail vein 72 hours after feeding. During the experiment, all groups of rats were provided with sufficient food and free access to water.

[0138] In this embodiment, the control rat diet was a homemade rat diet containing only bovine whey protein (20%). The arginine-restricted homemade rat diet contained 20% recombinant protein powder. Information on the corresponding recombinant proteins in the arginine-restricted homemade rat diet is shown in the table below: Table 25. Information on recombinant proteins in arginine-restricted rat diets

[0139] The preparation method of recombinant protein dry powder is shown in Example 9.

[0140] Blood was collected from the tail vein of rats on days 0 and 3. Immediately after collection, the blood was placed on ice and centrifuged at 4°C to collect plasma. All collected plasma samples were numbered, labeled, and stored at -80°C. Once all samples were collected, the plasma was serially diluted 10-100 times using an arginine detection kit (enzyme-linked immunosorbent assay), and the arginine content was measured according to the manufacturer's instructions. During the 3-day experiment, observations of the rats' food intake, excretion, fur, movement, and body weight showed that rats fed a specially formulated recombinant protein powder diet had normal food and water intake and no significant adverse reactions.

[0141] There were no significant differences in baseline plasma arginine levels among all groups of rats, with an average of 31.2 ± 2.68 mg / L. In the control group fed a diet containing bovine whey protein, plasma arginine levels did not change significantly during the experiment (Day 3: 32.2 ± 1.75 mg / L, p>0.05). In rats fed a diet containing arginine-restricted recombinant protein powder, plasma arginine levels decreased to 4.73–11.56 mg / L on Day 3, a reduction of 62.9%–84.8%. Furthermore, the lower the arginine content in the recombinant protein fed to the rats, the higher the degree of plasma arginine depletion. The table below illustrates the results using the three groups with the lowest, highest, and moderate arginine content in the protein powder used: Table 26. Arginine content in rat plasma (mg / L)

[0142] SDS-PAGE electrophoresis results of recombinant proteins ArgR1-1, ArgR2-1, and ArgR4-1 are shown in the figure. Figure 1 .

[0143] The results show that the arginine-free protein powder produced by the technology of this invention can effectively deplete the arginine content in rat plasma in a short period of time. Furthermore, the higher the purity of the prepared protein powder and the lower the arginine content, the better the depletion effect. In the examples described, the arginine content was consistently below 10.0 mg / g, all showing significant effects.

[0144] Example 9: Feeding mice with arginine-free / low-arginine protein powder can inhibit the growth of breast cancer xenografts. In this embodiment, recombinant protein powders with low, medium, and high arginine contents were used to prepare rat food to illustrate their beneficial effects in the animal model. Referring to the results in Example 7, the representative recombinant proteins selected in this embodiment are: ArgR1-1 (low), ArgR2-1 (medium), and ArgR4-1 (high).

[0145] The experimental animals used in this embodiment were 6-week-old Balb / c mice weighing 20 ± 2.0 g, housed in an SPF-grade animal facility. After one week of acclimatization to the environment with normal mouse feed and distilled water, all mice were randomly divided into groups of 5, with each group housed in a single cage. All mice in both groups received human breast cancer MDA-MB-231 cells, specifically: human breast cancer MDA-MB-231 cells were cultured in vitro until adhesion reached 70-90%. The cells were then seeded at 1.0 × 10⁻⁶ cells per cell line. 8 MDA-MB-231 cells were injected into the two groups of mice at a concentration of 0.1 mL / mL, with each mouse receiving the cells via the axilla. The day of injection was recorded as day 0.

[0146] From day 1 until the end of the experiment, mice in different groups were fed different specially formulated rat diets (the rat diet preparation method is the same as in Example 8), as follows: ① Bovine whey protein group: Feed them homemade rat food containing only bovine whey protein (20%).

[0147] ②PAR1 protein powder group (low): From day 1 to the end of the experiment, the rats were fed homemade rat food containing only ArgR1-1 protein powder (20%).

[0148] ③PAR2 protein powder group (medium): From day 1 to the end of the experiment, the mice were fed homemade rat food containing only ArgR2-1 protein powder (20%).

[0149] ④PAR3 Protein Powder Group (High): From day 1 to the end of the experiment, the rats were fed homemade rat food containing only ArgR4-1 protein powder (20%).

[0150] During the experiment, the mice were not restricted in their food supply and were allowed free access to water.

[0151] The size of the tumor was measured using vernier calipers, and the diameter of the tumor was measured with calipers every 5 days. The results are shown in […]. Figure 2 The amount of food consumed by the mice was measured daily, and the average value was calculated. The results are shown below. Figure 3 Mice body weight was measured on day 0 and day 40, and the results are shown in [the table below]. Figure 4 On day 40, the experiment ended and all mice were euthanized. Blood samples were taken on day 0 and day 40 to determine the arginine content (μmol / L) in the plasma. The results are shown in the table below.

[0152] Table 27. Arginine content in mouse plasma (mg / L)

[0153] The results showed that, compared with the control group, the plasma arginine levels in mice in the PAR1, PAR2, and PAR3 groups were significantly lower (reduced by 58%–79%), and this decrease persisted for a relatively long period (~6 weeks). Consistent with Example 8, in this animal model, the lower the arginine content in the recombinant protein, the higher the degree of arginine consumption. Tumors (>5mm) in mice in the bovine whey protein (control) group... 3 The tumors appeared on day 15, while those in the PAR1, PAR2, and PAR3 groups appeared on days 30, 27, and 26, respectively. At the end of the experiment, compared to the control group, tumor growth was significantly inhibited in all mice whose diets were restricted to arginine. Figure 2 The changes in food intake and body weight of the two groups of mice were observed. The data of the PAR1, PAR2, and PAR3 groups were similar to those of the control group. Figure 3 and Figure 4 This indicates that the recombinant protein rat diet had no significant adverse effects on the model animals in this embodiment.

[0154] Example 10: Feeding mice with arginine-free / low-arginine protein powder can inhibit joint inflammation. In this embodiment, recombinant protein powders with low, medium, and high arginine contents were used to prepare rat food to illustrate their beneficial effects in the animal model. Referring to the results in Example 8, the representative recombinant proteins selected in this embodiment were: ArgR1-1 (low), ArgR2-1 (medium), and ArgR4-1 (high). The experimental animals in this embodiment were male C57BL / 6 mice aged ~8 weeks and weighing 15 ± 1.5 g. After acclimatizing to the environment for 1 week by feeding them normal rat food and drinking distilled water, they were randomly divided into groups of 5 mice each. The modeling method was the same for mice in different groups, using a type II collagen-induced arthritis model. The specific process was as follows: type II collagen was dissolved in acetic acid, stirred thoroughly at 4°C until completely dissolved, and left at 4°C overnight. A complete Freund's adjuvant was prepared, and the complete Freund's adjuvant was mixed with the type II collagen solution taken at 4°C at a 1:1 volume ratio and emulsified to prepare a type II collagen emulsion. On day 1, 0.1 mL of this emulsion was injected intradermally into the base of the mouse tail. On day 21, mice were injected intraperitoneally with 0.1 mL of type II collagen emulsion. Joint swelling was assessed and scored on day 42. Mouse weight was measured on days 0 and 42. On day 0, blood was collected from the orbital sinus, and the serum was stored at -80°C. Mice were anesthetized and euthanized at the end of the experiment on day 42, and blood was collected from the heart; the serum was stored at -80°C.

[0155] From day 1 until the end of the experiment, mice in different groups were fed different specially formulated rat diets (the rat diet preparation method is the same as in Example 8): ① Whey protein group: Feed them homemade rat food containing only whey protein.

[0156] ②PAR1 protein powder group (low): From day 1 to the end of the experiment, the mice were fed homemade rat food containing only ArgR1-1 protein powder.

[0157] ③PAR2 protein powder group (medium): From day 1 to the end of the experiment, the mice were fed homemade rat food containing only ArgR2-1 protein powder.

[0158] ④PAR3 Protein Powder Group (High): From day 1 to the end of the experiment, the mice were fed homemade rat food containing only ArgR4-1 protein powder.

[0159] During the experiment, the mice were not restricted in their food supply and were allowed free access to water.

[0160] On the same day, the arginine content in mouse serum was measured using a kit at day 0 and day 42.

[0161] Table 28. Serum arginine content in mice on day 42

[0162] The arthritis index was scored based on the degree, extent, and deformity of the joints in mice.

[0163] Table 29. Arthritis Index Scores of Mice on Day 42

[0164] The results showed that the serum arginine content of mice fed a specially formulated rat diet with arginine-restricted protein powder was effectively reduced, consistent with the results in Examples 8 and 9. In the animal model of this example, the lower the arginine content in the recombinant protein, the higher the degree of arginine consumption.

[0165] Observations showed that after the initial immunization, the local inflammatory response in the control group mice took about a week to heal, while the PAR1, PAR2, and PAR3 groups showed milder symptoms and recovered within 3-5 days. After a second immunization, on day 42, the total arthritis scores of the PAR1, PAR2, and PAR3 groups were significantly lower than those of the control group. Changes in mouse body weight recorded during the experiment are shown in [see attached table]. Figure 5 Apart from slight hair loss, no other adverse reactions were observed in any of the experimental groups of mice. It can be inferred that the hair loss reaction is related to inflammation and is unrelated to the recombinant protein prepared in this invention.

Claims

1. A recombinant dietary protein lacking arginine, characterized in that, The amino acid sequence of the recombinant dietary protein is shown in SEQ ID NO.

1.

2. A vector comprising a nucleic acid sequence encoding the recombinant dietary protein of claim 1.

3. A recombinant microorganism comprising the vector provided in claim 2.

4. The recombinant microorganism according to claim 3, characterized in that, The microorganism mentioned is a Bacillus licheniformis expression system.

5. The recombinant microorganism according to claim 4, characterized in that, The Bacillus licheniformis strain mentioned is strain 10266Δ9. Strain 10266Δ9 is a strain modified from CICC 10266 by knocking out or silencing 9 genes encoding extracellular secretory proteins, namely cat, epr, bpr1, bpr2, apr, yhfN, SpoIIAC, lytC, and mpr, resulting in a secretory protein-deficient strain.

6. A method for preparing the recombinant dietary protein of claim 1, the method comprising culturing the recombinant microorganism of any one of claims 3-5 under conditions suitable for producing dietary protein by said recombinant microorganism.

7. A composition comprising the arginine-free recombinant dietary protein of claim 1.

8. The use of the recombinant dietary protein of claim 1 or the composition of claim 7 in the preparation of products that reduce or control arginine levels in the body.

9. The use of the recombinant dietary protein of claim 1 or the composition of claim 7 in the preparation of a medicament for controlling and treating argininemia, arthritis or inhibiting breast cancer growth.

10. The use of the recombinant dietary protein of claim 1 or the composition of claim 7 in the preparation of special medical foods.

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