Polypeptide asparagine ligase VdiPAL1 and application thereof

Abstract

The invention discloses a polypeptide asparagine ligase VdiPAL1 and an application of the polypeptide asparagine ligase VdiPAL1. The polypeptide asparagine ligase VdiPAL1 is newly found from viola plants, 12 mg / L zymogen protein is obtained through expression of an escherichia coli prokaryotic system, and compared with homologous enzymes VyPAL2 and VdiPAL1 which are widely applied at present, the polypeptide asparagine ligase VdiPAL1 has the advantages of high expression, high catalytic efficiency and substrate specificity and has good development and application potential. The invention also shows the value of the VdiPAL1 for guiding homologous enzyme modification, and particularly discloses the mutant protein VyOPT of the polypeptide asparagine ligase VyPAL2 and a coding cDNA (complementary deoxyribonucleic acid) sequence of the mutant protein VyOPT. The invention also discloses a mutant VyOPT, which comprises five mutations obtained by modification guided by the VdiPAL1. Compared with the expression quantity of the natural VyPAL2, which is about 0.1 mg / L, the expression quantity of the VyOPT reaches 12 mg / L.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a polypeptide asparagine ligase VdiPAL1 and its uses. Background Technology

[0002] Peptide ligases are specialized enzymes that catalyze the formation of intramolecular and intermolecular peptide bonds in polypeptide or protein molecules. They are widely used in the synthesis, modification, coupling, and cyclization of peptides and proteins, and have significant research and application value. Compared to hydrolytic proteases that are ubiquitous in nature, peptide ligases with ligation activity are relatively rare in nature. Currently, their discovery mainly relies on natural cyclic peptides and related splicing products from plant sources, and their functional identification is based on their biosynthetic pathways.

[0003] Among them, peptide asparaginyl ligase (PAL) has become one of the most promising ligase types due to its extremely high catalytic efficiency, extremely low hydrolysis side reaction rate, and broad substrate adaptability. In 2014, Tam's research group first isolated and identified Butelase-1, a member of the PAL family, from butterfly pea (Clitoria ternatea), and their related research was included in the international patent WO 2015 / 163818 A1. Subsequently, in 2019, Hemu et al. used transcriptome sequencing to discover the highly efficient and low-hydrolysis PAL enzyme VyPAL2 from Viola yedoensis, and successfully applied it to multiple fields such as protein end modification and antibody-drug conjugates. From a sequence and structural perspective, PAL is highly similar to asparaginyl endopeptidase (AEP), both belonging to the C13 family of cysteine ​​proteases (legumain family), but their catalytic directions are opposite—PAL mediates peptide bond formation, while AEP performs hydrolysis. Based on structural analysis and mechanism exploration, Hemu et al. proposed the "LAD" (ligase activity determinants) hypothesis, pointing out that the conserved residue types at LAD1 and LAD2 sites can effectively distinguish between linkage-type PAL and hydrolytic-type AEP. The applications of VyPAL2 in peptide and protein synthesis and LAD in PAL identification have been covered by international patent WO 2020 / 226572A1.

[0004] The LAD hypothesis provides a theoretical basis for PAL sequence prediction and database mining, significantly improving the screening efficiency of PAL and enabling PAL discovery to gradually shift from traditional isolation and characterization to high-throughput mining based on computational prediction. In 2023, Hemu et al. screened 1500 plant legumain database sequences using the LAD rule, identifying 18 PALs with ligation activity, accounting for only 1% of homologous sequences, further verifying the rarity of this type of ligase in nature. Currently, known PAL sources mainly include cyclic peptide-containing plants such as *Butterfly Pea*, *Viola*, *Helianthus*, and *Helianthus*, with more than half originating from the *Viola* genus. *Viola* species are abundant and widely distributed geographically, with over two hundred species in China, and most of them naturally produce cyclic peptides, making them ideal natural plants for expanding the diversity of PAL ligases.

[0005] Currently, only ten classes of peptide ligases are known. Among them, PatG, GmPOPB, PCY1, and SurE are oligopeptide cyclases and cannot be effectively used for intermolecular ligation reactions. Trypsiligase and Connectase are only used for ligation reactions. Intein can only react with substrates in a one-to-one ratio via fusion expression. Only three ligation tools—Sortase series, subtiligase series, and PAL—possess multifunctional activity and are suitable for large-scale in vitro production. Sortase series have lower efficiency and leave residual pentapeptide scar sequences in the product; subtiligase series substrates require C-terminal esterification, affecting their biocompatibility. PAL has extremely high catalytic efficiency and very low hydrolytic side reactions, near-scarless ligation products, and a wide range of reaction conditions and substrates, making it the protein modification tool with the greatest application potential.

[0006] However, due to the limited number of natural PAL sequences and their lack of sequence diversity, known PAL sequences generally exhibit low expression levels in prokaryotic expression systems, poor thermal stability in alkaline environments, and their linkage is based on acyl transfer reactions, exhibiting a degree of reversibility. These shortcomings severely hinder the further development and industrial application of PAL. Therefore, expanding the natural plant-derived PAL sequence library and identifying new, advantageous PAL enzymes is of great value. Summary of the Invention

[0007] Objective of the Invention: This invention aims to discover a polypeptide asparagine ligase (PAL) with high expression levels and high catalytic efficiency by collecting and transcribing the transcriptomes of Viola plants rich in cyclic peptides. The technical problem to be solved by this invention is to screen and identify an excellent ligase VdiPAL1 from Viola dissecta, which exhibits good solubility and expression levels in prokaryotic expression systems, and has the ability to catalyze peptide bond formation reactions with high efficiency and novel substrate specificity.

[0008] Another technical problem to be solved by the present invention is to provide a nucleic acid molecule encoding the polypeptide asparagine ligase VdiPAL1.

[0009] Given the high sequence homology between VdiPAL1 and the widely used ligase VyPAL2, but their significant differences in expression performance in prokaryotic systems, this invention uses VdiPAL1 as a reference template to directionally modify the VyPAL2 sequence, thereby effectively increasing VyPAL2 expression. Therefore, this invention also aims to provide a mutant protein and mutant gene of the peptide asparagine ligase VyPAL2. This enzyme and its directed mutant variants can be applied to highly efficient targeted modification, coupling, or cyclization reactions of peptides and proteins, possessing significant research and application value.

[0010] Another technical problem that this invention aims to solve is to provide expression cassettes, recombinant vectors, recombinant cells, or recombinant bacteria.

[0011] Another technical problem to be solved by the present invention is to provide the application of the polypeptide asparagine ligase VdiPAL1, the nucleic acid molecule, the mutant protein, the mutant gene, the expression cassette, the recombinant vector, the recombinant cell or recombinant bacteria in catalytic reactions.

[0012] Technical solution: In order to solve the above technical problems, the present invention provides a polypeptide asparagine ligase VdiPAL1, wherein the amino acid sequence of the polypeptide asparagine ligase VdiPAL1 is as shown in SEQ ID NO.1 or SEQ ID NO.3 or has 90% or 95% or more identity with SEQ ID NO.1 or SEQ ID NO.3.

[0013] The protein crystal of the polypeptide asparagine ligase VdiPAL1 has a space group of P 21 21 2 and a unit cell parameter of P 21 21 2. α = 90°, β = 90°, γ = 90°, and the X-ray diffraction resolution is

[0014] The present invention also includes a nucleic acid molecule encoding the polypeptide asparagine ligase VdiPAL1, wherein the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.2 or SEQ ID NO.4 or has 70%, 80% or 90% or more identity with SEQ ID NO.2 or SEQ ID NO.4.

[0015] The present invention also includes a mutant protein of the polypeptide asparagine ligase VyPAL2, which is homologous to the VdiPAL1. The mutant protein is subjected to one or more of the following mutations on the polypeptide asparagine ligase VyPAL2: T201A, N162D, N119D, M357N, N397D, I351L, N356Q, M357K, N397E, A422D, and Q454R.

[0016] Preferably, the mutant protein of the polypeptide asparagine ligase VyPAL2 described in this invention is named recombinant VyOPT proenzyme, and the amino acid sequence of the recombinant VyOPT proenzyme is shown in SEQ ID NO.5.

[0017] The present invention also includes a mutant gene of the polypeptide asparagine ligase VyPAL2, wherein the mutant gene encodes a mutant protein of the mutant polypeptide asparagine ligase VyPAL2, and preferably, its nucleotide sequence is shown in SEQ ID NO.6.

[0018] The present invention also includes expression cassettes, recombinant vectors, recombinant cells or recombinant bacteria, which contain the nucleic acid molecules or the mutant genes described herein.

[0019] The present invention also includes the application of the aforementioned polypeptide asparagine ligase VdiPAL1, the aforementioned nucleic acid molecule, the aforementioned mutant protein, the aforementioned mutant gene, the aforementioned expression cassette, recombinant vector, recombinant cell, or recombinant bacteria in catalytic reactions. Preferably, the catalytic reaction includes a substrate cyclization reaction or a reaction linking a substrate to a nucleophilic peptide.

[0020] The substrate includes polypeptide GL12, the amino acid sequence of which is GIARGDYKSNSL, the linker is KALYINQL, and the amino acid sequence of the nucleophilic peptide is GXKRY.

[0021] Preferably, the catalytic reaction includes a catalytic linking reaction between an affibody protein and a fluorescent peptide substrate. The affibody protein includes affibody-NQL, and the fluorescent peptide substrate is GISK(FAM)K(N3)-COOH.

[0022] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: This invention newly discovered a polypeptide asparagine ligase, VdiPAL1, from Violets. Through expression in the Escherichia coli prokaryotic system, a zymogen protein level of 12 mg / L was obtained, far superior to the expression level of the homolog VyPAL2 (VyPAL2 expression level in the E. coli prokaryotic system is approximately 0.1 mg / L). Compared with the widely used homolog VyPAL2, VdiPAL1 combines the advantages of high expression, high catalytic efficiency, and substrate specificity, showing good potential for development and application. Simultaneously, based on its sequence, the homolog VyPAL2 was modified, ultimately obtaining a mutant strain VyOPT with 5 mutation sites, increasing the expression level from approximately 0.1 mg / L to 12 mg / L. Attached Figure Description

[0023] Figure 1 This is a diagram of the VdiPAL1 expression plasmid; the VdiPAL1 expression gene is 1449 bp, and the total plasmid is 6658 bp.

[0024] Figure 2 The figure shows the amino acid logo diagram of the PAL and non-PAL sequences in legumain. As shown in the figure, the PAL and non-PAL sequences are significantly different in the LAD2, S2', LAD1 and MLA regions, which can be used as screening criteria.

[0025] Figure 3 SDS-PAGE gel image for VdiPAL1 purification. Figure 3 From left to right in A: zymogen purified by Ni column, with a target molecular weight of 55 kDa; unpurified core domain after acidification (32 kDa); core domain purified by acidification (32 kDa); Marker; Figure 3 From left to right in B are: 55kDa proenzyme purified from SEC; Marker;

[0026] Figure 4 SDS-PAGE gel images of purified glutamine cyclase QC and affibody-NQL proteins; Figure 4 Figure A shows affibody protein and marker purified by Ni column, with a target molecular weight of 9.7 kDa; Figure B shows QC protein and marker with SUMO tag purified by Ni column, with a target molecular weight of 55 kDa.

[0027] Figure 5 The graph shows the difference in reactivity of VdiPAL1 at different pH values, indicating that it has a wide range of reaction conditions, with the optimal reaction pH being 6.5.

[0028] Figure 6The figure shows the comparison of enzyme kinetics between VdiPAL1 and VyPAL2; VdiPAL1 (Km = 4.14 μM, kcat = 1.83 s) -1 kcat / Km = 442029M -1 s -1 The catalytic efficiency is superior to that of the widely used VyPAL2 (Km = 5.77 μM, kcat = 1.81 s⁻¹). -1 kcat / Km = 313692M -1 s -1 );

[0029] Figure 7 The effect of nucleophilic peptide P2” residue differences on VdiPAL1 ligation reaction is shown in the figure; the substrate peptide KALYINQL (200 μM) and the affinity peptide GXKRY (1 mM) were ligated, and 200 nM VdiPAL1 ligase and QC were added. The conversion rate of the ligation product was detected by liquid chromatography.

[0030] Figure 8 This is a schematic diagram of the reaction between affibody and fluorescent substrate peptide; the final concentrations of the protein substrate affibody-NQL and the fluorescent peptide GISK(FAM)K(N3)-COOH are 1 mM and 2 mM, respectively, and the final concentrations of VdiPAL1 and QC are 1 μM. The reaction is carried out in a phosphate buffer at pH 6.5; the substrate and product are marked with arrows in the liquid phase diagram, and "*" represents impurities in the substrate;

[0031] Figure 9 The image shows the protein crystal structure of VdiPAL1.

[0032] Figure 10 Figure showing the sequence alignment results of VyPAL2 and VdiPAL1;

[0033] Figure 11 This is a diagram showing the structural alignment results of VyPAL2 and VdiPAL1; purple represents the VyPAL2 protein, and white represents the VdiPAL1 protein.

[0034] Figure 12 Screening diagram of fluorescence signals for VyPAL2 single mutations;

[0035] Figure 13 This is a screening diagram of combined mutation fluorescence signals in traditional iterations of VyPAL2.

[0036] Figure 14 A computationally assisted screening diagram of combined mutation fluorescence signals for VyPAL2. Detailed Implementation

[0037] Example 1: Molecular cloning to obtain the VdiPAL1 sequence

[0038] Transcriptome sequencing was performed on collected Viola diffusa plants (Wenchuan, Sichuan). The sequencing results were analyzed using blastp analysis with the VyPAL2 amino acid sequence (Uniprot ID A0A509GV09) as a template to screen for a novel PAL enzyme, named VdiPAL1. Simultaneously, Viola diffusa plant mRNA was extracted and reverse transcribed to obtain cDNA, which was used as a cloning template. Primer pairs were designed based on the sequencing results (forward primer: 5'-CTGGTGCCGCGCGGCAGCCATCTCCGGTTGCCGTCAGAAG-3'; reverse primer: 5'-GTGGTGGTGGTGGTGGTGCTCGAGCGCACTGAACCCTTCGG-3'). PCR was performed with multiple rounds of pre-denaturation (95℃, 3 min), denaturation (95℃, 15 s), annealing (61℃, 15 s), and extension (72℃, 90 s) to obtain the target sequence. The pET28a(+) vector was double-digested with NcoI and XhoI (Takara). The VdiPAL1 gene was then homologously recombined into the digested pET28a(+) vector using the ClonExpress II One Step Cloning Kit (Novizan), and transformed into E. coli. In DH5α, positive clones were selected by PCR identification and confirmed to be correct by sequencing using universal primers T7 (5'-TAATACGACTCACTATAGG-3') and T7 terminator (5'-GCTAGTTATTGCTCAGCGG-3'). Thus, the recombinant plasmid pET28a(+)-VdiPAL1 shown in SEQ ID NO.4 was obtained.

[0039] In summary, the asparagine polypeptide ligase VdiPAL1 gene of this invention is derived from the plant cDNA of Viola diffusa. Figure 1 The amino acid sequence of the protein encoding the asparagine polypeptide ligase VdiPAL1 is shown in SEQ ID NO. 1 or SEQ ID NO. 3, and the nucleotide sequence is shown in SEQ ID NO. 2 or SEQ ID NO. 4. Sequence alignment of the newly discovered VdiPAL1 with the previously discovered legumain, Seq Logo analysis shows that the PAL sequence is highly conserved in the LAD2, S2', and MLA regions related to the catalytic direction, while the corresponding regions in the non-PAL sequence show relatively low overall conservation. Figure 2 ).

[0040] Example 2: VdiPAL1 protein expression and purification

[0041] The pET28a(+)-VdiPAL1 recombinant plasmid obtained in Example 1 was transformed into competent E. coli shuffle T7 strain (Shanghai Weidi Biotechnology Co., Ltd.). The plasmid was added to competent cells, incubated on ice for 30 min, heat-shocked at 42°C for 1 min, then cultured at 37°C for 1 h after adding LB liquid medium, and plated overnight at 37°C. Transformants were picked and cultured overnight at 37°C to obtain the fermentation seed culture. 1% (v / v) of fresh seed culture was inoculated into 3L shake flasks containing 1L of LB liquid medium and cultured at 37°C until OD (dryness). 600 Once the pH reached 0.6, IPTG was added to a final concentration of 0.1 mM for induction. Cells were cultured at 16°C and 180 rpm for 48 h on a shaker, followed by centrifugation to harvest the cells. Cells were resuspended at a ratio of 10 mL / g in lysis buffer (50 mM HEPES, 0.1 M NaCl, 5% glycerol, 5 mM β-mercaptoethanol, 1 mM EDTA, 1 mM PMSF, 0.1% Triton X-100, pH 7.5), lysed by sonication, and then centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was collected and purified by nickel affinity chromatography using an AKTA start FPLC system (Cytiva) equipped with a HisTrap HP column to obtain inactive zymogen, i.e., the complete core domain-linker-cap domain form. The expression level of the zymogen protein was 12 mg / L, significantly better than the expression level of VyPAL2 protein in the *E. coli* system (approximately 0.1 mg / L). The VdiPAL1 protein expressed in this embodiment of the invention is a recombinant protein obtained by removing the signal peptide and modifying the amino acid sequence of the natural asparagine polypeptide ligase VdiPAL1 as shown in SEQ ID NO.1. The amino acid sequence of the recombinant VdiPAL1 protein is shown in SEQ ID NO.3.

[0042] Example 3: Acidification, Activation, and Purification of VdiPAL1

[0043] The inactive prozyme purified by the Ni column in Example 2 was acidified to a final pH of 4.5 with a protein concentration of 0.5 mg / mL using an acidification buffer (20 mM sodium citrate, 0.1 M NaCl, 5 mM β-mercaptoethanol, 1 mM EDTA). The mixture was then acidified in a 37°C water bath for 10 minutes, resulting in the self-activation of the prozyme into active core and cap domains. The acidified protein was further concentrated by ultrafiltration at 4000 g and 4°C. Purification was then performed using an SEC gel column, separating the core and cap domains based on molecular weight. The core was collected by ultrafiltration to a concentration of 1 mg / mL, yielding a pure enzyme with an active core (purity >90%). SDS-PAGE results are shown below. Figure 3 A. After acidification and purification, a core domain protein band of the same size as the target was obtained. Simultaneously, the VdiPAL1 prozyme, purified by Ni column, was purified by SEC gel column, and the acidified protein was concentrated by ultrafiltration at 4000g and 4℃. The SDS-PAGE results are as follows. Figure 3 B, the target molecular weight of the zymogen is 55kDa, and the purity is >90%, which can be used for protein crystallization screening.

[0044] Example 4: Expression and purification of recombinant glutamine cyclase QC and recombinant affibody-NQL protein

[0045] The recombinant affibody-NQL protein gene (gene sequence shown in SEQ ID NO.7) and recombinant glutamine cyclase QC (gene sequence shown in SEQ ID NO.8) were synthesized by Genscript Biotech Co., Ltd. NcoI and XhoI restriction sites were selected and cloned into the pET28a(+) expression vector to obtain expression plasmids. The amino acid sequences of the recombinant expressed proteins are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively. Transformations were performed on E. coli BL21(DE3) competent cells (Shanghai Weidi Biotechnology Co., Ltd.). Transformants were picked and cultured overnight at 37°C in LB broth to serve as fermentation seed cultures. 1% (v / v) of fresh fermentation seed culture was inoculated into 3L shake flasks containing 1L of LB broth and cultured at 37°C until OD. 600 Once the pH reached 0.6, IPTG was added to a final concentration of 0.1 mM for induction. The cells were cultured at 16°C and 180 rpm for 48 h on a shaker, followed by centrifugation to harvest the cells. Cells were resuspended at a ratio of 10 mL / g in lysis buffer (50 mM HEPES, 0.1 M NaCl, 5% glycerol, 5 mM β-mercaptoethanol, 1 mM EDTA, 1 mM PMSF, 0.1% Triton X-100, pH 7.5), lysed by sonication, and then centrifuged at 12000 rpm for 30 min at 4°C. The supernatant was collected and analyzed using a HisTrap column. The affibody-NQL protein and the glutamine cyclase QC with a solubilization tag (SUMO-hQC) were purified by nickel affinity chromatography using an FPLC system (Cytiva). Their SDS-PAGE results are shown below. Figure 4 A and Figure 4 As shown in B, the molecular weight of the affibody-NQL protein is approximately 9.7 kDa, which is consistent with the expected size, and the size of the SUMO-hQC is approximately 55 kDa, which is also consistent with the expected size.

[0046] Example 5: Effect of different pH values ​​on VdiPAL1 activity

[0047] After obtaining the core domain of the purified enzyme VdiPAL1 in Example 3, enzyme properties were characterized. The model substrate polypeptide GL12 (GIARGDYKSNSL, synthesized by Nanjing Jietai Biotechnology Co., Ltd.) was selected for cyclization activity testing at different pH values. The enzyme concentration was 20 nM, and the substrate concentration was 20 μM. The reaction was conducted in a reaction buffer at pH 4.5–8 (citrate or phosphate, specifically pH 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8), at 37°C for 10 minutes in a water bath, followed by heat quenching. The substrate conversion rate was detected by HPLC. The results are shown in [link to relevant documentation]. Figure 5 ,from Figure 5 It can be seen that the optimal reaction pH for the VdiPAL1 protein of this invention is 6.5. This property is similar to that of PAL in the genus Violets.

[0048] Example 6: Kinetics of VdiPAL1 and VyPAL2 Cycloning Reaction

[0049] To evaluate the catalytic efficiency of the enzyme, cyclization reaction kinetics were performed at the optimal pH 6.5. The VdiPAL1 and VyPAL2 proteins prepared in Example 3 (VyPAL2 protein was prepared using the exact same method as in Examples 2 and 3) were subjected to cyclization reaction kinetics at the optimal pH 6.5. Different concentrations of GL12 substrate peptide (2.5, 5, 10, 20, 30, and 40 μM) and 10 nM of purified enzyme were added to the reaction system. The different substrate concentrations were reacted in a water bath at 37°C for 30, 60, and 90 s, respectively. Substrate conversion was detected by HPLC after quenching. Results are shown below. Figure 6 As shown, from Figure 6 It can be seen that VdiPAL1 (Km=4.14μM, kcat=1.83s) -1 kcat / Km = 442029M -1 s -1 The catalytic efficiency is superior to that of the widely used VyPAL2 (Km = 5.77 μM, kcat = 1.81 s⁻¹). -1 kcat / Km = 313692M -1 s -1 ).

[0050] Example 7: Effect of differences in nucleophilic peptide P2” residues on VdiPAL1 linker response

[0051] Next, the specificity of the nucleophilic substrate P2” for the ligation reaction of this enzyme was evaluated. The ligation specificity was tested using the ligation substrate KALYINQL (200 μM) synthesized from Nanjing Jietai Biotechnology Co., Ltd., and the nucleophilic peptide P2”: GXKRY (1 mM), where X represents 20 different amino acids. VdiPAL1 prepared in Example 3 with a final concentration of 200 nM and recombinant glutamine cyclase QC prepared in Example 4 with a final concentration of 200 nM were reacted in 20 mM phosphate buffer (pH 6.5) at 37 °C for 30 min, followed by quenching at 100 °C for 5 min. The conversion rate of the cyclization product was detected by HPLC. The results are as follows: Figure 7 Ultimately, it was found that when X is Trp, Phe, Met, Ile, or Leu, the linkage yield reaches over 90%, with X = Trp being the optimal choice. This is significantly different from the optimal residues at the same site in VyPAL2, which are Leu / Ile.

[0052] Example 8: VdiPAL1-catalyzed affibody and fluorescent peptide substrate linkage reaction

[0053] Previous systematic studies on the optimal pH conditions and substrate specificity of VdiPAL1 have verified that this enzyme possesses excellent peptide cyclization and coupling catalytic capabilities. To further evaluate the application potential of VdiPAL1 in protein drug construction, this example uses the engineered small protein Affibody as a model to conduct its fluorescent labeling modification reaction. Affibody, as a novel antibody substitute molecule, has good structural stability, high affinity, excellent tissue penetration, and low immunogenicity, and has been widely used in tumor targeted therapy and molecular imaging probes. Affibody-NQL protein was prepared according to the method in Example 4, and the substrate peptide GISK(5-FAM)K(N3)-COOH (synthesized by Nanjing Jietai Biotechnology Co., Ltd.) containing the 5-FAM fluorescent group was synthesized. Under the synergistic effect of the glutamine cyclase (QC) synthesized in Example 4, VdiPAL1 can efficiently catalyze the directional linkage between 1 mM affibody and 2 mM peptide substrate at physiological temperature (37°C). After 90 minutes of reaction, the substrate was completely converted into the target product, demonstrating the high efficiency of VdiPAL1 enzymatic ligation and its green reaction characteristics under mild conditions. This one-pot enzymatic chemical reaction system provides an efficient and simple ligation strategy for the construction of protein drugs containing small-molecule functionalized peptides, and is particularly suitable for developing molecular probes and therapeutic agents targeting extracellular tumor-related targets such as epidermal growth factor receptor (EGFR).

[0054] The specific steps of this embodiment are as follows: The substrate affibody-NQL and the fluorescent peptide GISK(FAM)K(N3)-COOH were added to the reaction system at final concentrations of 1 mM and 2 mM, respectively. VdiPAL1 prepared in Example 3 and recombinant glutamine cyclase QC prepared in Example 4, both at final concentrations of 1 μM, were added. The ligation reaction was carried out in a phosphate buffer at 37°C and pH 6.5. Samples were taken at 30, 60, and 90 minutes for HPLC detection. The results are as follows: Figure 8 After 30 minutes of reaction, the substrate conversion rate was about 50%, and after 90 minutes of reaction, the substrate was almost completely converted into the linker product.

[0055] Example 9: Crystal structure screening of VdiPAL1

[0056] To improve the prokaryotic expression performance of VyPAL2, this invention uses VdiPAL1 as a reference for design mutations. Therefore, obtaining the crystal structure information of VdiPAL1 helps to understand the key information of the differential residues more deeply.

[0057] Therefore, in this embodiment, the purified VdiPAL1 proenzyme obtained in Example 3 was concentrated to 17 mg / ml. A 96-well droplet screening and condition optimization were performed using a HamptonIndex HT (Hampton Research, HR2-144, USA) crystallization kit. Cubic protein crystals were grown in a crystallization buffer (0.2 M ammonium sulfate, 0.1 M HEPES, pH 7.7, 27% w / v PEG 3350). Structural data were obtained by X-ray diffraction, and structural analysis was performed using CCP4i and PHENIX software. The structure was further refined using COOT software. Figure 9 The final resolution obtained is The high-quality dimer crystal structure is shown in Table 1, and the crystal structure parameters are shown in Table 1. Through structural comparison, VdiPAL1 and VyPAL2 (PDB ID 6IDV) show extremely high overall similarity (RMSD = 0.74). Figure 11 ).

[0058] Table 1. Data collection and structure optimization parameters for VdiPAL1 crystal.

[0059]

[0060]

[0061] Example 10: VyPAL2 Expression Modification Guided by VdiPAL1

[0062] VdiPAL1 and VyPAL2 both originate from the genus Violets and are on the same evolutionary branch in the phylogenetic tree, exhibiting high sequence and structural similarity. Figure 10 and Figure 11 However, the expression performance varies significantly in prokaryotic systems. VyPAL2, as one of the most thoroughly studied peptide asparagine ligases, has an extremely low rate of hydrolytic side reactions, making it an ideal ligase tool. However, its expression level in E. coli is extremely low (approximately 0.1 mg / L), often requiring expensive insect cell systems for expression, which severely limits its widespread application in industry or research.

[0063] Next, sequence alignment of VdiPAL1 and VyPAL2 revealed only 30 differing residues. These 30 differentially expressed sites were identified, and based on this, 30 single-point mutants of VyPAL2 were designed and constructed. Each mutation replaced the corresponding VyPAL2 site with a residue from VdiPAL1 to systematically evaluate the impact of these differentially expressed sites on expression levels. Specifically, the fluorescence signals were sequenced after mutating VyPAL2 to the sites on VdiPAL1. Figure 12 To avoid affecting catalytic activity, the first 7 residues located in the cap (M357, N356, N397, I351, A422, V409, Q454) domain were selected as hotspots.

[0064] To achieve high-throughput screening, we introduced a GFP fusion protein expression system. Previous validation showed a positive correlation between GFP fluorescence intensity and PAL protein expression levels, making it a potential surrogate indicator of expression levels. Fluorescence intensity was measured in 30 GFP fusion expression monomutators at 480 nm excitation and 520 nm emission wavelengths. 22 of these mutants showed higher fluorescence signals than the wild-type VyPAL2, with the M357N mutation being the most significant, exhibiting a fluorescence intensity 1.4 times that of the wild-type. Further analysis revealed that among the top ten most expressed mutations, only T201A, N162D, and N119D were located in the core domain, while the remaining seven were located in the cap domain, mostly at the terminal turns of α-helical or loop structures. It is speculated that these residues may indirectly affect overall expression performance by regulating the folding stability of the cap structure. Based on these screening results, to further improve expression levels and avoid impacting the activity of the catalytic core domain, this invention selected the seven dominant monomutators located in the cap domain for combined mutation. First, a greedy iterative strategy is adopted. Next, a traditional method is used for combination, starting with M357N as the starting material (M1). Double mutations are then performed with the remaining 6 residues (M2), with the strongest fluorescence signal observed in the double mutation M357N+N397D. Next, triple mutations are performed with the remaining 5 residues (M3), with the optimal mutation set being M357N+N397D+Q454R. This process continues until the fluorescence signal in the M5 mutant does not exceed that of M4, reaching the endpoint. The optimal mutant is M357N+N397D+Q454R+I351L in M4. Figure 13 When GFP expression was removed, the expression level reached 7 mg / L.

[0065] Since the expression levels of the optimal combination mutations obtained through traditional iterations did not exceed those of VdiPAL1, a computational method was chosen to guide the combination. Initially, for 30 differentially expressed sites, individual mutation screening identified 7 dominant mutations. For the 7 sites corresponding to these 7 mutations, all site combinations with sampling numbers of 2, 3, 4, and 5 were obtained. A cyclic mutation design was performed using the Protein_MPNN protein language model with a temperature parameter of 0.1, and predicted combinations containing at least 40% dominant mutations were selected as candidates, resulting in 265 preliminary candidate combinations. Subsequently, Rosetta_ddg was used to calculate the free energy change (ΔΔG) of all 265 candidate mutation combinations, and 25 combinations with ΔΔG values ​​below -1.5 kcal / mol were selected for experimental validation. Experimental results showed that... Figure 14Among them, the fluorescence signals of C3 (I351L+V409P+N356R+A422D) and C18 were higher than those of VdiPAL1. The optimal five mutant C18 (I351L / N356Q / M357K / N397E / A422D) was de-GFP expressed and the expression level reached the highest 12 mg / L in the E. coli prokaryotic expression system (method as in Example 2), which is about 120 times higher than that of wild type, thus obtaining the optimal mutation.

Claims

1. A polypeptide asparagine ligase VdiPAL1, characterized in that, The amino acid sequence of the polypeptide asparagine ligase VdiPAL1 is as shown in SEQ ID NO.1 or SEQ ID NO.3, or has 90% or 95% or more of the same identity as SEQ ID NO.1 or SEQ ID NO.

3.

2. The polypeptide asparagine ligase VdiPAL1 according to claim 1, characterized in that, The protein crystal of the polypeptide asparagine ligase VdiPAL1 has space group P 21 21 2, cell parameters a = 115.31 Å, b = 128.90 Å, c = 70.96 Å, α = 90°, β = 90°, γ = 90°, and X-ray diffraction resolution of 1.87 Å.

3. A nucleic acid molecule encoding the polypeptide asparagine ligase VdiPAL1 as described in claim 1 or 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.2 or SEQ ID NO.4, or has 70%, 80%, or 90% or more of the same identity as SEQ ID NO.2 or SEQ ID NO.

4.

4. A mutant protein of VyPAL2, a polypeptide asparagine ligase homologous to VdiPAL1 as described in claim 1, characterized in that, The mutant protein has one or more of the following mutations on the peptide asparagine ligase VyPAL2: T201A, N162D, N119D, M357N, N397D, I351L, N356Q, M357K, N397E, A422D, and Q454R. Preferably, the amino acid sequence of the mutant protein is shown in SEQ ID NO.

5.

5. A mutant gene for the polypeptide asparagine ligase VyPAL2, characterized in that, The mutant gene encodes a mutant protein of the mutant polypeptide asparagine ligase VyPAL2 as described in claim 4. Preferably, the mutant gene sequence is shown in SEQ ID NO.

6.

6. An expression cassette, a recombinant vector, a recombinant cell, or a recombinant bacterium, characterized in that, It contains the nucleic acid molecule as described in claim 3 or the mutant gene as described in claim 5.

7. The use of the polypeptide asparagine ligase VdiPAL1 of claim 1 or 2, the nucleic acid molecule of claim 3, the mutant protein of claim 4, the mutant gene of claim 5, the expression cassette of claim 6, the recombinant vector, the recombinant cell or recombinant bacteria in catalytic reactions.

8. The application according to claim 7, characterized in that, The catalytic reaction includes a catalytic substrate cyclization reaction or a reaction linking a substrate to a nucleophilic peptide, wherein the substrate includes a polypeptide GL12, the amino acid sequence of which is GIARGDYKSNSL, the linking substrate is KALYINQL, and the amino acid sequence of which is GXKRY.

9. The application according to claim 7, characterized in that, The catalytic reaction includes a reaction that catalyzes the linking of affibody protein and fluorescent peptide substrate.

10. The application according to claim 9, characterized in that, The affibody protein is affibody-NQL, and the fluorescent polypeptide substrate is GISK(FAM)K(N3)-COOH.

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