A catalytic antibody mutant degrading helicobacter pylori urease and its preparation method and application

Abstract

The application belongs to the field of biological medicine, and particularly relates to a catalytic antibody mutant for degrading Helicobacter pylori urease, and a preparation method and application thereof. The application takes wild-type catalytic antibody Ua15-L (WT) for degrading Helicobacter pylori urease as a starting antibody, rationally designs a catalytic antibody mutant for degrading Helicobacter pylori urease with better performance, and verifies through experiments that the I48C-G64C mutant is mutated at the 48th and 64th amino acids, and is mutated from isoleucine and glycine to cysteine, so that a disulfide bond is formed between the two, which is helpful to improve the thermal stability of the catalytic antibody. On this basis, the mutations L9E, L54R and T14Q-I15P are introduced to increase the protein solubility and expression amount. Compared with the wild-type catalytic antibody for degrading Helicobacter pylori urease, the mutant 1 (L9E-I48C-G64C) increases the Tm value by 6 DEG C, increases the solubility by 81%, and increases the expression amount by 4 times; and the time for the urease fragment to be hydrolyzed by half at 37 DEG C is shortened by 52% compared with the wild type.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a catalytic antibody mutant that degrades Helicobacter pylori urease, its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] A catalytic antibody is an antibody that not only binds to a specific antigen but also catalyzes the corresponding chemical reaction. The first catalytic antibody was discovered in the plasma of a patient with bronchial asthma in 1989; subsequently, numerous catalytic antibodies have been found in individuals with various diseases or in healthy individuals. Furthermore, catalytic antibodies can be acquired through immunization with immunogens or transition state analogs. The effects of catalytic antibodies can be further enhanced through directed evolution or rational design.

[0004] Helicobacter pylori is the only bacterium that can survive in the highly acidic environment of the human stomach. Numerous studies have confirmed that Helicobacter pylori is a major cause of chronic gastritis, gastric ulcers, and duodenal ulcers, and is closely related to the development of gastric cancer. The light chain UA15-L of the catalytic antibody can cleave the peptide bonds of Helicobacter pylori urease, thereby inhibiting the spread of Helicobacter pylori. The variable region (VL) of the light chain performs protease functions, while the constant region (CL) helps maintain solubility and thermal stability and therefore does not directly function. Removing the constant region of the light chain can reduce the protein size by half, thereby effectively increasing the drug potency of the same protein mass and providing an advantage in drug delivery. Compared to the intact antibody or the entire light chain, the VL of UA15-L (i.e., UA15-VL) exhibits relatively poor thermal stability and solubility, which hinders its therapeutic application. Therefore, enhancing the thermal stability and solubility of this catalytic antibody is necessary. Summary of the Invention

[0005] To overcome the above problems, the present invention provides a catalytic antibody mutant that degrades Helicobacter pylori urease, its preparation method and application.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a catalytic antibody mutant that degrades Helicobacter pylori urease, wherein the mutant is mutated at any one or more sites selected from the group consisting of:

[0008] L9E, T14Q, I15P, I48C, G64C, L54R;

[0009] The amino acid residue numbers are based on SEQ ID NO.1 (the amino acid sequence of the wild-type catalytic antibody against Helicobacter pylori urease (Ua15-L(WT)), MGSSHHHHHHSSGLVPRGSHMDVVMTQTPLTLSVTIGQPASISCKSSQSLLDS DGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK).

[0010] The catalytic antibody mutant for degrading Helicobacter pylori urease is mutated based on the wild-type catalytic antibody for degrading Helicobacter pylori urease described above, and the catalytic antibody mutant for degrading Helicobacter pylori urease is selected from the following group of mutants:

[0011] Mutant 1: L9E-I48C-G64C; (amino acid sequence as shown in SEQ ID NO.2);

[0012] The amino acid sequence shown in SEQ ID NO.2 is as follows:

[0013] MGSSHHHHHHSSGLVPRGSHMDVVMTQTPETLSVTIGQPASISCKSSQSL LDSDGKTYLNWLLQRPGQSPKRLCYLVSKLDSGVPDRFTCSGSGTDFTLKISR VEAEDLGVYYCWQGTHFPQTFGGGTKLEIK.

[0014] Mutant 2: L9E-T14Q-I15P-I48C-G64C; (amino acid sequence as shown in SEQ ID NO.3);

[0015] The amino acid sequence shown in SEQ ID NO.3 is as follows:

[0016] MGSSHHHHHHSSGLVPRGSHMDVVMTQTPETLSVQPGQPASISCKSSQS LLDSDGKTYLNWLLQRPGQSPKRLCYLVSKLDSGVPDRFTCSGSGTDFTLKIS RVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK.

[0017] Mutant 3: L9E-I48C-G64C-L54R. (Amino acid sequence as shown in SEQ ID NO.4);

[0018] The amino acid sequence shown in SEQ ID NO.4 is as follows:

[0019] MGSSHHHHHHSSGLVPRGSHMDVVMTQTPETLSVTIGQPASISCKSSQSL LDSDGKTYLNWLLQRPGQSPKRLCYLVSKRDSGVPDRFTCSGSGTDFTLKISR VEAEDLGVYYCWQGTHFPQTFGGGTKLEIK.

[0020] In a second aspect, the present invention provides a polynucleotide encoding a catalytic antibody mutant for degrading Helicobacter pylori urease as described in the first aspect.

[0021] A third aspect of the present invention provides a recombinant expression vector containing the polynucleotides described in the second aspect above.

[0022] In a fourth aspect, the present invention provides a host cell containing the vector described in the third aspect or a chromosome integrated with the polynucleotide described in the second aspect or expressing a catalytic antibody mutant that degrades Helicobacter pylori urease as described in the first aspect.

[0023] A fifth aspect of the present invention provides a method for preparing the above-mentioned catalytic antibody mutant that degrades Helicobacter pylori urease, comprising:

[0024] The host cells described in the fourth aspect above are cultured to express the catalytic antibody mutant that degrades Helicobacter pylori urease; and the catalytic antibody mutant that degrades Helicobacter pylori urease is isolated and purified.

[0025] A sixth aspect of the present invention provides the use of the catalytic antibody mutant that degrades Helicobacter pylori urease as described in the first aspect in the preparation of a medicament for improving, preventing or treating Helicobacter pylori infection.

[0026] A seventh aspect of the present invention provides a medicament for improving, preventing or treating Helicobacter pylori infection, comprising the catalytic antibody mutant that degrades Helicobacter pylori urease as described in the first aspect.

[0027] An eighth aspect of the present invention provides a pharmaceutical composition comprising the catalytic antibody mutant for degrading Helicobacter pylori urease as described in the first aspect and one or more pharmaceutically acceptable carriers.

[0028] The beneficial effects of this invention are as follows:

[0029] This invention provides a mutant catalytic antibody for degrading Helicobacter pylori urease, its preparation method, and its application. Specifically, this invention uses a wild-type catalytic antibody for degrading Helicobacter pylori urease as the starting antibody. Using bioinformatics and protein engineering techniques, a mutant catalytic antibody for degrading Helicobacter pylori urease is obtained. Experimental verification shows that in the I48C-G64C mutant, amino acids at positions 48 and 64 are mutated, from isoleucine and glycine to cysteine, respectively, forming a disulfide bond between them. This helps improve the thermal stability of the catalytic antibody; however, this mutation significantly reduces the solubility and expression level of the catalytic antibody. Based on this, additional mutations L9E, L54R, and T14Q-I15P are introduced to increase protein solubility and, hopefully, restore protein expression without sacrificing thermal stability. Compared to the wild-type catalytic antibody for degrading Helicobacter pylori urease, mutant 1L9E-I48C-G64C increases the Tm by 6℃, increases solubility by 81%, and increases expression level by 4 times. The mutant 1L9E-I48C-G64C also showed better urease fragment hydrolysis activity at 37°C compared to the wild type. Attached Figure Description

[0030] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0031] Figure 1 To map Tmp to the 3D structure of UA15-VL;

[0032] Figure 2 The effective contact area between I48 and G64 varies with temperature;

[0033] Figure 3 The thermal stability of Ua15-L(WT) and its mutants is defined as follows: A is Ua15-L(WT), B is mutant 4I48C-G64C, C is mutant 1L9E-I48C-G64C, D is mutant 2L9E-T14Q-I15P-I48C-G64C and E is mutant 3L9E-I48C-G64C-L54R.

[0034] Figure 4The figures represent the proteolytic activities of Ua15-L(WT) and its mutants. A represents the hydrolytic activity of Ua15-L(WT) at 25℃, B represents the hydrolytic activity of Ua15-L(WT) at 37℃, C represents the hydrolytic activity of mutant 1L9E-I48C-G64C at 25℃, D represents the hydrolytic activity of mutant 1L9E-I48C-G64C at 37℃, and E represents the hydrolytic activity of mutant 2L9E- The hydrolytic activity of T14Q-I15P-I48C-G64C at 25℃, F represents the hydrolytic activity of mutant 2L9E-T14Q-I15P-I48C-G64C at 37℃, G represents the hydrolytic activity of mutant 3L9E-I48C-G64C-L54R at 25℃, and H represents the hydrolytic activity of mutant 3L9E-I48C-G64C-L54R at 37℃. Detailed Implementation

[0035] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0037] A first typical embodiment of the present invention provides a catalytic antibody mutant that degrades Helicobacter pylori urease, wherein the mutant is selected from any one or more sites of the following group:

[0038] L9E, T14Q, I15P, I48C, G64C, L54R;

[0039] The amino acid residue numbers are based on SEQ ID NO.1 (the amino acid sequence of the wild-type catalytic antibody against Helicobacter pylori urease (Ua15-L(WT)), MGSSHHHHHHSSGLVPRGSHMDVVMTQTPLTLSVTIGQPASISCKSSQSLLDS DGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK).

[0040] The catalytic antibody for degrading Helicobacter pylori urease described in this invention contains only the light chain UA15-L of the Ua15 catalytic antibody and does not contain the heavy chain of the catalytic antibody for degrading Helicobacter pylori urease. Studies have shown that adding the heavy chain of the Ua15 catalytic antibody does not provide any activity for degrading Helicobacter pylori urease.

[0041] The catalytic antibody mutant for degrading Helicobacter pylori urease is mutated based on the wild-type catalytic antibody for degrading Helicobacter pylori urease described above, and the catalytic antibody mutant for degrading Helicobacter pylori urease is selected from the following group of mutants:

[0042] Mutant 1: L9E-I48C-G64C; (amino acid sequence as shown in SEQ ID NO.2);

[0043] The amino acid sequence shown in SEQ ID NO.2 is as follows:

[0044] MGSSHHHHHHSSGLVPRGSHMDVVMTQTPETLSVTIGQPASISCKSSQSL LDSDGKTYLNWLLQRPGQSPKRLCYLVSKLDSGVPDRFTCSGSGTDFTLKISR VEAEDLGVYYCWQGTHFPQTFGGGTKLEIK.

[0045] Mutant 2: L9E-T14Q-I15P-I48C-G64C; (amino acid sequence as shown in SEQ ID NO.3);

[0046] The amino acid sequence shown in SEQ ID NO.3 is as follows:

[0047] MGSSHHHHHHSSGLVPRGSHMDVVMTQTPETLSVQPGQPASISCKSSQS LLDSDGKTYLNWLLQRPGQSPKRLCYLVSKLDSGVPDRFTCSGSGTDFTLKIS RVEAEDLGVYYCWQGTHFPQTFGGGTKLEIK.

[0048] Mutant 3: L9E-I48C-G64C-L54R. (Amino acid sequence as shown in SEQ ID NO.4);

[0049] The amino acid sequence shown in SEQ ID NO.4 is as follows:

[0050] MGSSHHHHHHSSGLVPRGSHMDVVMTQTPETLSVTIGQPASISCKSSQSL LDSDGKTYLNWLLQRPGQSPKRLCYLVSKRDSGVPDRFTCSGSGTDFTLKISR VEAEDLGVYYCWQGTHFPQTFGGGTKLEIK.

[0051] Preferably, in mutants 1-3, the amino acids at positions 48 and 64 are mutated, with isoleucine and glycine respectively mutating to cysteine, and a disulfide bond is formed between them.

[0052] In mutants 1-3, amino acids at positions 48 and 64 are mutated, with isoleucine and glycine respectively mutating to cysteine, and a disulfide bond is formed between them. The construction of the disulfide bond helps to improve the thermal stability of the catalytic antibody that degrades Helicobacter pylori urease.

[0053] Mutants 1L9E-I48C-G64C, 2L9E-T14Q-I15P-I48C-G64C, and 3L9E-I48C-G64C-L54R can increase the solubility of the catalytic antibody that degrades Helicobacter pylori urease.

[0054] Mutants 1L9E-I48C-G64C, 2L9E-T14Q-I15P-I48C-G64C, and 3L9E-I48C-G64C-L54R can increase the expression level of catalytic antibodies that degrade Helicobacter pylori urease.

[0055] The mutant 1L9E-I48C-G64C can enhance the activity of the catalytic antibody that degrades Helicobacter pylori urease.

[0056] A second typical embodiment of the present invention provides a polynucleotide encoding a catalytic antibody mutant for degrading Helicobacter pylori urease as described in the first aspect.

[0057] A third typical embodiment of the present invention provides a recombinant expression vector containing the polynucleotides described in the second aspect above.

[0058] In one or more embodiments, the recombinant expression vector is obtained by effectively linking the aforementioned polynucleotide to an expression vector. The expression vector is any one or more of a viral vector, plasmid, bacteriophage, kinase, or artificial chromosome. The viral vector may include an adenovirus vector, a retrovirus vector, or an adeno-associated virus vector. The artificial chromosome includes a bacterial artificial chromosome, a vector derived from bacteriophage P1, a yeast artificial chromosome, or a mammalian artificial chromosome. Preferably, the expression vector is a plasmid. In one specific embodiment of the present invention, the plasmid is pET28a.

[0059] A fourth typical embodiment of the present invention provides a host cell containing the vector described in the third aspect above or a chromosome integrated with the polynucleotide described in the second aspect above or expressing the catalytic antibody mutant that degrades Helicobacter pylori urease described in the first aspect above.

[0060] In one or more embodiments, the host cell may be a prokaryotic cell or a eukaryotic cell.

[0061] Preferably, the host cell is any one or more of bacterial cells and fungal cells;

[0062] The bacterial cells mentioned therein are any species within the genera Escherichia, Agrobacterium, Bacillus, Streptomyces, Pseudomonas, or Staphylococcus;

[0063] More preferably, the bacterial cells are Escherichia coli (such as Escherichia coli BL21(DE3)), Agrobacterium tumefaciens (such as GV3101), Agrobacterium rhizogenes, Bacillus subtilis, Bacillus cereus, or Pseudomonas.

[0064] The fungal cells include yeasts (such as Pichia pastoris).

[0065] A fifth typical embodiment of the present invention provides a method for preparing the above-mentioned catalytic antibody mutant that degrades Helicobacter pylori urease, comprising:

[0066] The host cells described in the fourth aspect above are cultured to express the catalytic antibody mutant that degrades Helicobacter pylori urease; and the catalytic antibody mutant that degrades Helicobacter pylori urease is isolated and purified.

[0067] The sixth typical embodiment of the present invention provides the use of the catalytic antibody mutant that degrades Helicobacter pylori urease as described in the first aspect above in the preparation of a medicament for improving, preventing or treating Helicobacter pylori infection.

[0068] A seventh typical embodiment of the present invention provides a medicament for improving, preventing or treating Helicobacter pylori infection, comprising the catalytic antibody mutant that degrades Helicobacter pylori urease as described in the first aspect.

[0069] An eighth typical embodiment of the present invention provides a pharmaceutical composition comprising the catalytic antibody mutant for degrading Helicobacter pylori urease as described in the first aspect and one or more pharmaceutically acceptable carriers.

[0070] The pharmaceutically acceptable carrier may be a diluent, excipient, filler, binder, humectant, disintegrant, absorption enhancer, adsorbent, surfactant, or lubricant, but is not limited thereto.

[0071] The pharmaceutical composition is used to improve, prevent or treat Helicobacter pylori infection.

[0072] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0073] Example 1

[0074] Mutant 4: I48C-G64C (amino acid sequence shown in SEQ ID NO. 5). Amino acids at positions 48 and 64 are mutated, replacing isoleucine and glycine with cysteine, respectively. Using the genome of strain Ua15-L(WT) as a DNA template, PCR amplification was performed using primer pairs I48C-F / I48C-R and G64C-F / G64C-R, respectively.

[0075] I48C-F: 5'-TGTTATCTGGTTAGCAAACTGGATAGCGG-3';

[0076] I48C-R:5'-CAGACGTTTA GGGCTCTGAC CTG-3'.

[0077] G64C-F: 5'-TGTAGCGGCAGCGGTACCGATTT-3';

[0078] G64C-R: 5'-GGTAAAACGATCTGGCACAC CGC-3'.

[0079] Mutant 4: The I48C-G64C amino acid sequence (shown in SEQ ID NO.5) is as follows:

[0080] MGSSHHHHHHSSGLVPRGSHMDVVMTQTPLTLSVTIGQPASISCKSSQSL LDSDGKTYLNWLLQRPGQSPKRLCYLVSKLDSGVPDRFTCSGSGTDFTLKISR VEAEDLGVYYCWQGTHFPQTFGGGTKLEIK.

[0081] Mutant 1: L9E-I48C-G64C; (amino acid sequence as shown in SEQ ID NO.2); using the genome of strain I48C-G64C as a DNA template, PCR amplification was performed on L9E-F / L9E-R using primer pairs respectively:

[0082] 9E-F:5'-GAAACCCTGAGCGTGACAATTGGTC-3';

[0083] 9E-R:5'-CGGCGTCTGGGTCATAACAACAT-3'.

[0084] Mutant 3: L9E-I48C-G64C-L54R (amino acid sequence shown in SEQ ID NO.4); Mutant 1: L9E-I48C-G64C underwent a mutation at amino acid position 54, changing from leucine to arginine. Using the genome of strain L9E-I48C-G64C as a DNA template, PCR amplification was performed using primer pairs L54R-F and L54R-R respectively.

[0085] L54R-F: 5'-CGTGATAGCGGTGTGCCAGATCGTTTTAC-3';

[0086] L54R-R: 5'-TTTGCTAACCAGATAACACAGACGTTTAGGG-3'.

[0087] Mutant 2: L9E-T14Q-I15P-I48C-G64C; (amino acid sequence shown in SEQ ID NO.3); Mutant 1: L9E-I48C-G64C underwent mutations at amino acid positions 14 and 15, replacing threonine and isoleucine with glutamine and proline, respectively. Using the genome of strain L9E-I48C-G64C as a DNA template, PCR amplification was performed using primer pairs T14Q-I15P-F / T14Q-I15P-R:

[0088] T14Q-I15P-F: 5'-CAGCCGGGTCAGCCGGCAAGCATTAGTTG-3';

[0089] T14Q-I15P-R: 5'-CACGCTCAGG GTTCCGGC-3'.

[0090] The PCR amplification system consisted of 25 μL of 2×Pimer Star Max DNA mixed enzyme solution, 20 ng of DNA template, 1 μL each of primers (10 μM), and 22.5 μL of distilled water, for a total volume of 50 μL.

[0091] The amplification conditions were as follows: 98℃ pre-denaturation for 3 minutes (1 cycle); 98℃ denaturation for 10 seconds, 70℃ annealing for 15 seconds, and 72℃ extension for 2 minutes and 30 seconds (20 cycles), with the annealing temperature decreasing by 0.5℃ per cycle; 72℃ extension for 3 minutes and 30 seconds (1 cycle); 98℃ denaturation for 10 seconds, 66℃ annealing for 15 seconds, and 72℃ extension for 2 minutes and 30 seconds (13 cycles); and 72℃ extension for 3 minutes and 30 seconds (1 cycle).

[0092] PCR amplification products were digested with restriction endonucleases Nco I and Xho I, and ligated with plasmid pET28a digested with the same enzymes. The ligation product was transformed into DH5α competent cells, incubated on ice for 30 minutes, heat-shocked at 42°C for 90 seconds, immediately placed on ice for 5 minutes, and then incubated with 500 μL of LB liquid medium at 200 rpm at 37°C for 1 hour. The transformed cells were plated on LB solid medium plates containing kanamycin (final concentration 50 μg / mL) and cultured overnight at 37°C. Four single colonies were picked and sent for sequencing. The expression plasmid with correct sequencing was transformed into E. coli BL21(DE3), successfully constructing the mutant expression engineered bacteria.

[0093] WT and all mutants were recombinantly expressed and purified in the same manner, as follows:

[0094] Fresh colonies were inoculated into 1 L LB medium containing 100 g / mL Kansat resistance (Kan) and cultured at 37°C in a shaker (approximately 220 rpm) until the OD600 reached 0.6–0.8. Then, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added to induce recombinant protein expression for 24 hours at 18°C. The induced bacterial culture was transferred to a centrifuge bottle and centrifuged at 2600 g, 4°C for 30 minutes. The culture was then resuspended in an appropriate amount of binding buffer, sonicated to lyse, and centrifuged at 13000 rpm, 4°C for 30 minutes. The supernatant was then filtered through a 0.45 μm pore size filter. Subsequently, the protein was expressed using a pre-equilibrated Ni-NTA column. Purification was performed using a pure chromatography system (GE Healthcare, Chicago, USA). Non-target proteins bound to the column were washed with buffer (20 mM Tris, 300 mM NaCl, pH 8.0). Finally, the target protein was eluted using a linear gradient of imidazole (20–500 mM). The solution was concentrated to less than 5 mL using an ultrafiltration tube with a molecular weight cutoff of 3 kDa, and then passed through a pre-equilibrated Superdex 75 molecular sieve column (Cytiva, Shanghai, China). Further purification was performed using a pure chromatography system (GE Healthcare, Chicago, USA). The buffer solution was 20 mM Tris, 80 mM NaCl, pH 8.0. Protein purity was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein concentration was measured using a Nanophotometer NP80 Microvolume spectrophotometer (Implen, Germany). The extinction coefficient of the purified protein was 17210 M. -1 cm -1 280nm. Store at -20℃.

[0095] Example 2

[0096] Since the three-dimensional structure of the catalytic antibody UA15-VL was unavailable, alphafold2 was used to predict protein structure models from its sequence. Most protein regions exhibited high predicted local distance difference test (pLDDT) scores, indicating high accuracy in overall structure prediction, except for C-terminal residues, which showed low pLDDTs, suggesting poor prediction for these residues. Although the global melting temperature is typically obtained experimentally during protein folding-unfolding, MD simulations at multiple temperatures can provide information about protein unfolding hotspots, described by local melting temperatures. Figure 1 The lower local melting temperature indicates that the region is unstable and therefore tends to defold first as the temperature increases.

[0097] A series of MD simulations were performed on UA15-VL within a temperature range of 23.2 °C (250 K) to 326.8 °C (600 K), with two independent MD runs (15 ns each) at each temperature. If two residues are separated by more than four amino acids in the sequence and their nearest distance in the starting structure is less than [missing value], [the simulations were performed]. If two residues are considered to be in contact (between heavy atoms), then they are considered to be in contact. A total of 207 contact points were identified. The distance between the C atoms of each pair of contacting residues was recorded at each temperature, and the average value was calculated. Due to unfolding, this distance generally increases at higher temperatures. The distance is converted into effective contact ce, and the contact is quantitatively compared using the equation ce = exp(r / r0), where r is the distance between the two C atoms, and r0 is a constant. This equation ensures that effective contact decreases with increasing distance, and the change in ce becomes smaller for even larger distances. ce is obtained by averaging over two runnable MD snapshots. <ce>, where <> denotes ensemble average. Then, the effective contact at different temperatures is scaled to ces(Ti) =<ce(Ti)> / <ce(T0)> ,in<ce(T0)> This is the average contact at the lowest temperature (23.2°C) in the MD simulation. Therefore, ces equals 1 at the lowest MD temperature. In this work, the average contact at four lowest temperatures is used. <ce>The value is used as the denominator to minimize numerical noise. Cess is plotted against temperature and fitted to an sigmoid function. Figure 2 ).

[0098]

[0099] Here, a, b, and c are three fitting parameters, where a corresponds to the ces value at the lowest temperature, b reflects the rate of decrease in ces, and c is the temperature at which ces equals a / 2. The temperature at which ces = 0.5 is defined as the local melting temperature Tmp of the contact pair. If the ces of a pair is greater than 0.5 even at the highest simulated temperature (326.8℃), its Tmp cannot be accurately extracted. Nevertheless, the value of 326.8℃ is assigned to the Tmp of the pair, corresponding to the lower limit of the residue pair. The Tmps of different residue pairs are listed in Table 1. To simplify the data, the Tmp of the pair is evenly distributed among the two constituent residues. If a residue forms multiple contact pairs, the lowest Tmp is selected for that residue. The residue-specific Tmp is mapped to the 3D structure of the enzyme to directly examine the weaknesses ( Figure 1 ).

[0100] Figure 1 To map Tmp to the 3D structure of UA15-VL. Figure 1 In the diagram, as Tmp increases, the color gradient changes from red through white to blue. Regions with lower Tmp values ​​tend to expand first at higher temperatures. Unconnected residues are shown in gray. Residues involved in the designed mutations are labeled, and their side chains are represented as bars. In addition, three complementarity-determining regions (CDRs) are labeled.

[0101] Most hotspots are located at or near flexible CDRs (Kabat numbers: CDR1, K24N34; CDR2, L50S56; and CDR3, W89T97) and have low Tmp, which is not surprising. However, CDRs are crucial for the functional realization of UA15-VL. Therefore, mutations in CDRs should be avoided to prevent perturbation of protein function. As shown in Table 1 (and... Figure 1 As shown, after excluding residues in the CDR region, the I48G64 pair has the lowest Tmp. In other words, it initiates the unfolding of the UA15-VL framework region (FR) according to MD simulations. By enhancing the interactions in this hotspot, contact can theoretically be maintained at high temperatures.

[0102] Table 1. Tmps for different residue pairs

[0103]

[0104]

[0105]

[0106] Local melting temperatures (Tmp) for different residue pairs. Residue pairs containing CDRs are shown in italics, while other residue pairs are shown in normal font.

[0107] Example 3

[0108] Designing disulfide bonds to improve the thermal stability of antibodies catalyzed by Helicobacter pylori urease degradation:

[0109] Disulfide bonds are a simple interaction that can prevent local unwinding. DbD2 predicted several potential disulfide bonds, including C3-C26, C6-C99, C14-C17, C23-C88, C32-C91, C34-C49, and C48-C64. The C23-C88 disulfide bond is already present in WT UA15-VL. Disulfide bonds C3-C26, C32-C91, and C34-C49 involve residues in CDR1, with the nucleophilic catalytic residue S27a located at its position. Mutations in these regions may impair antibody function. The C14-C17 disulfide bond connects two residues that are too close together. The C6-C99 disulfide bond is far from the hotspot, and only the C48-C64 disulfide bond is located in the I48-G64 hotspot region. To examine whether the C48-C64 disulfide bond truly enhances protein thermal stability, Ua15-L (WT) and mutant 4I48C-G64C protein samples were prepared. Their melting temperatures were measured using differential scanning calorimetry (DSC). Ua15-L (WT) showed two melting temperatures, Tm1 = 41.5 ± 0.3 °C and Tm2 = 53.2 ± 0.7 °C. Figure 3 (A). The mutant I48C-G64C also showed two melting temperatures, Tm1 = 51.1 ± 0.4℃ and Tm2 = 57.9 ± 0.3℃. Figure 3 (Figure B) indicates that disulfide bonds increased Tm1 and Tm2 by 9.6 °C and 4.7 °C, respectively. The increase in melting temperature suggests that the additional disulfide bonds help stabilize the protein and prevent it from unfolding at high temperatures.

[0110] Example 4

[0111] Adjusting the solubility and expression level of the antibody catalyzing the degradation of Helicobacter pylori urease:

[0112] During the preparation of the catalytic antibody for Helicobacter pylori urease, turbidity was observed in the protein solution when it was concentrated using a concentrator. The solubility of Ua15-L (WT) in a buffer of 20 mM Tris, 80 mM NaCl (pH 7.0) was approximately 1.1 ± 0.1 mg / mL, which was relatively low (Table 2). The introduction of the I48C-G64C mutation further reduced the solubility to 0.4 ± 0.1 mg / mL. High protein solubility is a prerequisite for potential therapeutic applications. To enhance protein solubility without sacrificing stability, a combination of the FoldX and CamSol methods was employed, with the former used for stability prediction and the latter for solubility prediction. This method was used to identify mutations (above I48C-G64C) that may have favorable solubility and stability properties, including L9E, L54R, T14Q, and I15P. First, the mutant 1L9E-I48C-G64C was tested. Its solubility was 2.0 ± 0.2 mg / mL, which was 4 times higher than that of the I48C-G64C mutant and 82% higher than that of Ua15-L (WT). The mutant 1L9E-I48C-G64C also showed two melting temperatures, Tm1 = 47.5 ± 0.6℃ and Tm2 = 63.6 ± 0.7℃. Figure 3 (C). Tm1 is about 3°C ​​lower than that of the I48C-G64C mutant, but Tm2 is about 6°C higher, indicating that the L9E mutation has a fairly complex effect on protein thermostability.

[0113] Starting with the L9E-I48C-G64C mutation, either the L54R or T14Q-I15P mutation was added. The solubility of mutant 2L9E-T14Q-I15P-I48C-G64C was 2.9 ± 0.4 mg / mL, approximately 2.7 times that of Ua15-L (WT) and 1.5 times that of mutant 1L9E-I48C-G64C, consistent with CamSol solubility predictions. However, the T14Q-I15P mutation reduced the melting temperature Tm1 to 42.8 ± 0.9 °C and Tm2 to 59.7 ± 0.5 °C. Figure 3 The L54R mutation (above L9E-I48C-G64C) increased protein solubility to 2.6 ± 0.3 mg / mL, compared to 2.0 ± 0.2 mg / mL for mutant 1 L9E-I48C-G64C. Measurements of protein thermal stability showed that mutant 3 L9E-I48C-G64C-L54R had only one melting temperature, Tm = 52.5 ± 0.2 °C, approximately 5 °C higher than the Tm1 of the L9E-I48C-G64C mutant and approximately 1 °C higher than the I48C-G64C mutant. Figure 3 (E).

[0114] Table 2. Solubility and expression levels of Ua15-L (WT) and its mutants.

[0115]

[0116] a This indicates the solubility measured at 4°C in a buffer solution of 20 mM Tris, 80 mM NaCl, and pH 7.0.

[0117] Besides protein thermal stability and solubility, high protein expression levels are beneficial for experimental purposes in drug development and future applications because they reduce the economic cost of drugs. The yield of wild-type UA15-VL prepared in *E. coli* was relatively low, producing only 0.1 mg of protein per liter of growth medium after purification. The double mutant I48C-G64C with an additional disulfide bond reduced the protein yield to 0.07 mg / L. The triple mutant L9E-I48C-G64C increased the protein yield to 0.5 mg / L. The tetra mutant L9E-I48C-G64C-L54R increased the yield to approximately 0.6 mg / L, while the penta mutant L9E-T14Q-I15P-I48C-G64C reduced the protein yield to approximately 0.3 mg / L. It appears that L9E-I48C-G64C and L9E-I48C-G64C-L54R have similar thermal stability (…). Figure 3 It exhibits more balanced protein properties in terms of solubility and expression (Table 2).

[0118] Example 5

[0119] The proteolytic activity of Ua15-L(WT) and its mutants:

[0120] Helicobacter pylori urease is a hexameric enzyme composed of six α-subunits and six β-subunits. UA15-VL targets the β-subunit and is able to cleave the peptide bond between Y241 and D242. A fluorescent protein ligand consisting of CFP-pep-YFP, where pep corresponds to the peptide sequence NHALDVADKYDVQVAIHTDT (from N232 to T251) in the urease β-subunit, was used to test the proteolytic activity of catalytic antibodies and their mutants that degrade Helicobacter pylori urease. Cleavage of the peptide bond reduced the resonance energy transfer (FRET) between CFP and YFP, resulting in increased cyan fluorescence and decreased yellow fluorescence. The cleavage reaction exhibited a biphasic reaction spectrum, a unique property of some catalytic antibodies. A sigmoid function was proposed to fit the reaction curves, yielding the parameter T1 / 2, which corresponds to the time point at which half of the substrate is converted to product. The T1 / 2 for Ua15-L (WT) at 25 °C was 1600 ± 30 min ( Figure 4 All three mutants, L9E-I48C-G64C, L9E-I48C-G64C-L54R, and L9E-T14Q-I15P-I48C-G64C, exhibited T1 / 2 values ​​comparable to Ua15-L (WT), indicating that the mutations had a minimal impact on catalysis. This is not surprising, as all mutation sites were carefully selected to be located far from the active site. In contrast, significantly larger changes were observed in the different mutants at 37 °C. The wild-type UA15-VL exhibited a T1 / 2 of 2170 ± 290 min, 36% higher than its activity at 25 °C, suggesting that the activity may be reduced due to its poorer thermostability (the activity measurement temperature was close to the protein's Tm1 of 41.5 °C). In contrast, mutant 1L9E-I48C-G64C reduced its T1 / 2 to 1050 ± 50 minutes, exhibiting approximately 46% higher efficiency than its activity at 25 °C, and almost twice the activity of the wild type at 37 °C, attributed to its improved thermostability. Mutant 2L9E-T14Q-I15P-I48C-G64C significantly increased its T1 / 2 to 3360 ± 300 minutes, while mutant 3L9E-I48C-G64C-L54R slightly increased its T1 / 2 to 1650 ± 350 minutes. Therefore, the activity measurements clearly demonstrate that mutant 1L9E-I48C-G64C exhibits superior performance compared to other mutants at 37 °C.

[0121] In this invention, we present a computational and experimental study to engineer catalytic antibodies that degrade Helicobacter pylori urease, resulting in antibodies with high thermal stability, high solubility, and enhanced expression. These properties are relevant to the development of protein drugs, extending shelf life, improving drug delivery, and reducing the cost of drug availability. To improve protein thermal stability, multi-temperature MD simulations were performed to identify hotspots in the catalytic antibodies that degrade Helicobacter pylori urease, and to improve protein thermal stability without interfering with protein function. Therefore, hotspots near CDRs and active sites, which may consist of the D1-H93-S27a catalytic triplet, should be avoided. With the aid of the DbD2 program, a disulfide bond was designed at one of the hotspots, which increased the protein melting temperature by 7–10 °C. Mutations significantly reduced protein solubility and expression. With the aid of the FoldX and CamSol methods, additional mutations L9E, L54R, and T14Q-I15P were introduced to increase protein solubility and hopefully restore protein expression without sacrificing thermal stability. The combined mutant L9E-I48C-G64C increased Tm1 by 6 °C, solubility by 81%, and expression level by 4-fold compared to Ua15-L(WT). This mutant showed proteolytic activity comparable to Ua15-L(WT) at 25 °C, but due to better stability, the time for the urease fragment to be hydrolyzed by half at 37 °C was 52% shorter than that of the wild type.

[0122] When optimizing protein solubility, it is important not to compromise protein thermal stability. FoldX was used to assess the effect of mutations on protein stability, while CamSol was used to assess their effect on solubility. Both methods provide a list of mutations that may enhance stability and solubility. All mutants tested experimentally increased protein solubility, but only L54R showed a slight improvement in protein thermal stability, indicating that the computational method is not very accurate in predicting stability. This computational strategy can be improved by including more methods for predicting protein thermal stability and solubility to generate a more reliable list of mutations for experimental validation.

[0123] In summary, we successfully identified a catalytic antibody mutant L9E-I48C-G64C that degrades Helicobacter pylori urease, which has enhanced thermal stability, solubility, expression level, and a 52% shorter time to hydrolyze the urease fragment at 37°C compared to the wild type.

[0124] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.< / ce> < / ce>

Claims

1. A catalytic antibody mutant that degrades Helicobacter pylori urease, characterized in that, The amino acid sequence of the catalytic antibody mutant that degrades Helicobacter pylori urease is shown in SEQ ID NO.

2.

2. A polynucleotide, characterized in that, The polynucleotide encodes the catalytic antibody mutant for degrading Helicobacter pylori urease as described in claim 1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector contains the polynucleotide as described in claim 2.

4. A host cell, characterized in that, The host cell contains the vector of claim 3 or a chromosome integrated with the polynucleotide of claim 2 or expressing the catalytic antibody mutant that degrades Helicobacter pylori urease of claim 1.

5. A method for preparing the above-mentioned catalytic antibody mutant for degrading Helicobacter pylori urease, characterized in that, include: The host cells of claim 4 are cultured to express the catalytic antibody mutant that degrades Helicobacter pylori urease; And to isolate and purify the catalytic antibody mutant that degrades Helicobacter pylori urease.

6. A drug for treating Helicobacter pylori infection, characterized in that, Includes the catalytic antibody mutant for degrading Helicobacter pylori urease as described in claim 1.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the catalytic antibody mutant for degrading Helicobacter pylori urease as described in claim 1, and one or more pharmaceutically acceptable carriers.

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