Peptide n-glycosidase f mutants and uses thereof
By optimizing the amino acid sequence of PNGase F, a peptide N-glycosidase F mutant was designed and expressed, which solved the problems of high production cost and long enzyme digestion time, achieved efficient enzyme digestion, reduced production cost and improved enzyme activity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- BEIJING TRANSGEN BIOTECH CO LTD
- Filing Date
- 2025-12-16
- Publication Date
- 2026-05-15
AI Technical Summary
The high production cost and complex production process of existing PNGase F limit its widespread application. Furthermore, the long digestion time of the regular and rapid versions affects its efficiency in glycoprotein research and analysis.
By mutating the 291st aspartic acid of wild-type PNGase F (which does not contain a signal peptide) to glycine, a peptide N-glycosidase F mutant was designed and expressed. Its amino acid sequence was optimized, and efficient expression and purification were achieved through recombinant plasmid and host cell modification, thereby reducing production costs.
Under the same conditions, the enzyme activity of the peptide N-glycosidase F mutant was significantly improved, reducing production costs. It also showed excellent enzymatic digestion effects in both standard and rapid buffer systems, promoting its application in various fields.
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Figure CN121653107B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology. More specifically, it relates to a peptide N-glycosidase F mutant and its applications. Background Technology
[0002] PNGase F is a glycoamide hydrolase derived from Gram-negative bacteria such as *Pseudomonas meningitidis*. It specifically and completely removes N-linked oligosaccharides, making it the most efficient enzymatic method for removing almost all N-linked glycan chains from glycoproteins. It plays a crucial role in glycoprotein research and analysis, including the study of oligosaccharides and the deglycosylation of glycoproteins. PNGase F can cleave high-mannose, hybrid, and complex oligosaccharides. Its cleavage site is the glycosidic bond between the innermost N-acetylglucosamine (GlcNAc) and asparagine residues, simultaneously converting the asparagine residues to aspartic acid.
[0003] The conventional PNGase F reaction typically requires a long time to complete. Currently, an optimized, rapid version of peptidyl-N-glycosidase F is available, capable of cleaving the sugar chain within minutes. Direct extraction of peptidyl-N-glycosidase F from *Pseudomonas meningitidis* is extremely time-consuming and costly, involving cumbersome steps and complex processes, with very low yields (approximately 0.1-0.5 mg of peptidyl-N-glycosidase F per liter of bacterial culture). Furthermore, *Pseudomonas meningitidis* is a pathogenic bacterium, unsuitable for large-scale fermentation production. These factors significantly limit the production, preparation, and application of peptidyl-N-glycosidase F. While the enzyme has been able to achieve heterologous recombinant expression in yeast or *Escherichia coli* in recent years, its relatively high price due to the complexity of production and purification processes limits its widespread application. Improving the activity of glycosidases and reducing production costs are among the problems that need to be addressed.
[0004] Therefore, there is a need to provide a peptide-N-glycosidase F with high activity and low production cost, which will promote its application in various fields. Summary of the Invention
[0005] One objective of this invention is to provide a mutant of peptide N-glycosidase F (PNGase F), whose enzyme activity is improved in both the standard and rapid cleavage systems, which is of great significance for optimizing enzyme performance and greatly reduces the production cost of PNGase F.
[0006] Another object of the present invention is to provide the application of the above-mentioned PNGase F mutant.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] The present invention first provides a peptide N-glycosidase F mutant, the amino acid sequence of which is shown in SEQ ID NO.1.
[0009] The present invention relates to a peptide N-glycosidase F mutant in which the aspartic acid at position 291 (corresponding to position 330 of the full-length sequence) of wild-type PNGase F without a signal peptide is mutated to glycine; wherein, the amino acid sequence of the wild-type PNGase F without a signal peptide is shown in SEQ ID NO.3, and its codon-optimized nucleotide sequence is shown in SEQ ID NO.4.
[0010] The nucleotide sequence encoding the above-mentioned peptide N-glycosidase F mutant is also within the scope of protection of this invention.
[0011] Furthermore, the nucleotide sequence encoding the above-mentioned peptide N-glycosidase F mutant is shown in SEQ ID NO.2.
[0012] Recombinant plasmids containing the above nucleotide sequences are also within the scope of protection of this invention.
[0013] In a specific embodiment of the present invention, the recombinant plasmid is pET28a-PNGaseF-D330G; pET28a-PNGaseF-D330G is obtained by inserting the nucleotide sequence encoding the peptide N-glycosidase F mutant, as shown in SEQ ID NO.2, between the NcoI and XhoI restriction sites of the pET28a plasmid, while keeping the other sequences of pET28a unchanged.
[0014] Recombinant cells containing the above-mentioned nucleotide sequences or recombinant plasmids are also within the scope of protection of this invention.
[0015] In a specific embodiment of the present invention, the host cell of the recombinant cell is a modified BL21 Escherichia coli.
[0016] In a preferred embodiment of the invention, the host cell is a BL21(DE3) competent cell.
[0017] The preparation method of the above-mentioned peptide N-glycosidase F mutant in this invention includes the following steps:
[0018] a1) Construct a recombinant plasmid pET28a-PNGaseF-D330G containing the nucleotide sequence shown in SEQ ID NO.2;
[0019] a2) The recombinant plasmid pET28a-PNGaseF-D330G was transformed into host cells BL21(DE3) competent cells, and expression was induced to obtain bacterial cells;
[0020] a3) The bacterial cells were broken, centrifuged, and the supernatant was obtained. After purification, the peptide N-glycosidase F mutant was obtained.
[0021] This invention further discloses the application of the above-mentioned peptide N-glycosidase F mutant, the above-mentioned nucleotide sequence, or the above-mentioned recombinant plasmid or recombinant cell in deglycosylation or the preparation of deglycosylated products.
[0022] In a specific embodiment of the present invention, the application can be in the deglycosylation process in a standard buffer system or a fast buffer system.
[0023] In a specific embodiment of the present invention, the deglycosylation is the removal of N-linked oligosaccharides from the glycoprotein.
[0024] In a specific embodiment of the present invention, the standard buffer system uses Glycoprotein Denaturing Buffer, GlycoBuffer 2 and NP-40 as buffer solutions.
[0025] In a specific embodiment of the present invention, the rapid version buffer system uses Rapid PNGase F Buffer.
[0026] The present invention further discloses a deglycosylation reagent or kit, wherein the reagent or kit contains the above-mentioned peptide N-glycosidase F mutant.
[0027] The beneficial effects of this invention are as follows:
[0028] The present invention relates to a peptide N-glycosidase F mutant (PNGase F mutant), which mutates the aspartic acid at position 291 of wild-type PNGase F (which does not contain a signal peptide) to glycine. Under the same conditions, the enzyme activity is significantly improved compared with competing products, and the enzyme activity is improved in both the ordinary buffer system and the rapid buffer system. This is of great significance for optimizing enzyme performance, greatly reduces the production cost of commercial PNGase F, and promotes the application of PNGase F in various fields. Attached Figure Description
[0029] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.
[0030] Figure 1 The SDS-PAGE results for the PNGase F mutant of this invention are shown.
[0031] Figure 2 This is a comparison of the enzyme activity of the PNGase F mutant and wild-type PNGase F in a standard buffer system.
[0032] Figure 3This is a comparison of the enzyme activity of the PNGase F mutant of the present invention and the competing product H1 in a standard buffer system.
[0033] Figure 4 This is a comparison of the enzyme activity of the PNGase F mutant and wild-type PNGase F in the rapid buffer system of the present invention.
[0034] Figure 5 This is a comparison of the enzyme activity of the PNGase F mutant of the present invention and the competing product H2 in a rapid buffer system. Detailed Implementation
[0035] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further explains the invention. Similar components in the drawings are indicated by the same reference numerals. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention. Many other methods exist in the prior art; the following embodiments provide a specific solution.
[0036] Example 1: Obtaining the PNGase F mutant with enhanced enzyme activity
[0037] By searching the Uniprot gene library and the PDB protein structure database, the wild-type PNGase F sequence without a signal peptide was obtained (shown in SEQ ID NO.3, with its codon-optimized nucleotide sequence shown in SEQ ID NO.4). Through multiple sequence alignment and structural analysis, combined with rational design, the aspartic acid at position 291 (corresponding to position 330 in the full length) of the amino acid sequence shown in SEQ ID NO.3 without a signal peptide was mutated to glycine. After heterologous protein expression and purification, the PNGase F mutant was finally obtained (the amino acid sequence is shown in SEQ ID NO.1, i.e., the PNGase F mutant without a signal peptide). The specific steps are as follows:
[0038] I. Construction of Recombinant Cells
[0039] The host-optimized nucleotide sequence of the PNGase F mutant (shown in SEQ ID NO.2) was synthesized by Nanjing Genscript Biotech Co., Ltd., and this nucleotide sequence was inserted into an expression vector and transformed into E. coli. Details are as follows:
[0040] The nucleotide sequence encoding the PNGase F mutant (shown in SEQ ID NO.2) was inserted between the NcoI and XhoI restriction sites of the pET28a plasmid, while keeping the other sequences of pET28a unchanged, to obtain the corresponding recombinant plasmid pET28a-PNGaseF-D330G; then the synthesized recombinant plasmid pET28a-PNGaseF-D330G was transformed into competent cells of Escherichia coli expression strain BL21(DE3), thereby constructing a recombinant Escherichia coli strain (i.e., recombinant bacteria) containing the recombinant plasmid pET28a-PNGaseF-D330G.
[0041] II. Expression and purification of PNGase F mutant
[0042] The recombinant bacteria were plated on kanamycin-resistant plates and incubated overnight at 37°C. The next day, single colonies were picked from the plates and inoculated into 10 mL of fresh LB medium, incubated overnight at 37°C and 220 rpm, and then inoculated into 1 L of LB medium and incubated at 37°C and 220 rpm until OD (digesterone) was reached. 600 The concentration was approximately 0.6-0.8, and IPTG was added to a final concentration of 0.5 mM. Expression was induced at 37℃ for 6 hours. The bacterial cells were collected by centrifugation, resuspended in lysis buffer, and the supernatant was collected after cell lysis. Affinity chromatography and ion exchange chromatography were then performed to collect the purified product, which was the PNGase F mutant.
[0043] The PNGase F mutant was dialyzed into Storage Buffer (20 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA, 50% Glycerol, pH 7.5). SDS-PAGE gel analysis results are shown below. Figure 1 As shown in the figure, the protein sample size obtained in this embodiment is 36 kDa, consistent with the expected size of the PNGase F mutant, indicating that the final protein sample obtained in this embodiment is the PNGase F mutant. Furthermore, sequencing confirmed its correctness, and its amino acid sequence is shown in SEQ ID NO.1.
[0044] Example 2: The PNGase F mutant exhibited higher enzyme activity than wild-type PNGase F and competing products in a standard buffer system.
[0045] The enzyme activity of the PNGase F mutant obtained in Example 1 in a standard buffer system was compared with that of wild-type PNGase F and competing products. The specific procedures are as follows:
[0046] 1. Add 1 μl of 10×Glycoprotein Denaturing Buffer (5% SDS, 400 mM DTT) to 1 μl of substrate RNase B (concentration of 2.5 μg / μl), and make up to 10 μl with deionized water;
[0047] 2. Incubate at 100℃ for 10 min to denature the substrate RNase B;
[0048] 3. Cool the glycoprotein on ice and centrifuge for 10 seconds;
[0049] 4. Add 2 μl of 10×GlycoBuffer 2 (500 mM Sodium Phosphate, pH 7.5), 2 μl of 10% NP-40 and 6 μl of H2O, for a total volume of 20 μl. Gently pipette to mix.
[0050] 5. Add 1 μl of serially diluted PNGase F mutant or wild-type PNGase F or competitor H1 (purchased from HanHai New Enzyme Biotechnology Co., Ltd., catalog number: HBP003005), and gently pipette to mix (the amount of PNGase F mutant added to the system is 20, 2, 0.2, and 0.02 ng, respectively; the amount of competitor H added is 20, 2, and 0.2 ng, respectively), and incubate at 37°C for 1 hour;
[0051] 6. SDS-PAGE analysis of enzyme digestion effect.
[0052] The enzyme digestion results of PNGase F mutant and wild-type PNGase F are as follows: Figure 2 As shown, when the amounts of PNGase F mutant and wild-type PNGase F added to the system are the same, the PNGase F mutant has a better enzymatic cleavage effect on the substrate RNase B than the wild-type PNGase F.
[0053] The enzymatic digestion results of PNGase F mutant and competitor H1 are as follows: Figure 3 As shown, when the amounts of PNGase F mutant and competitor H1 are the same in the system, PNGase F mutant has a better enzymatic cleavage effect on substrate RNase B than competitor H1.
[0054] Example 3: The PNGase F mutant exhibited higher enzyme activity than wild-type PNGase F or competing products in the rapid buffer system.
[0055] The enzyme activity of the PNGase F mutant obtained in Example 1 in the rapid buffer system was compared with that of wild-type PNGase F or a competing product. The specific procedures are as follows:
[0056] 1. Add deionized water to 1 μl of substrate RNase B (concentration of 2.5 μg / μl) to bring the total volume to 16 μl;
[0057] 2. Add 4 μl of 5×Rapid PNGase F Buffer (250 mM Sodium Phosphate (pH 7.5), 100 mM DTT, 2.5% SDS), bringing the total volume to 20 μl, and gently pipette to mix.
[0058] 3. Incubate at 80℃ for 2 minutes, then cool on ice;
[0059] 4. Add 1 μl of serially diluted PNGase F mutant or wild-type PNGase F competitor H2 (purchased from Hanhai New Enzyme Biotechnology Co., Ltd., catalog number HBP003006), gently pipette to mix (the amount of PNGase F mutant and competitor H added in the system is 80, 8, 0.8, and 0.08 ng, respectively), and incubate at 50℃ for 10 min;
[0060] 5. SDS-PAGE analysis of enzyme digestion effect.
[0061] The enzyme digestion results of PNGase F mutant and wild-type PNGase F are as follows: Figure 4 As shown, when the amounts of PNGase F mutant and wild-type PNGase F added to the system are the same, the PNGase F mutant has a better enzymatic cleavage effect on the substrate RNase B than the wild-type PNGase F.
[0062] The enzymatic digestion results of PNGase F mutant and competitor H2 are as follows: Figure 5 As shown, when the amounts of PNGase F mutant and competitor H2 are the same in the system, PNGase F mutant has a better enzymatic cleavage effect on substrate RNase B than competitor H2.
[0063] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.
Claims
1. A peptide N-glycosidase F mutant, characterized in that, The amino acid sequence of the peptide N-glycosidase F mutant is shown in SEQ ID NO.
1.
2. A nucleotide molecule encoding the peptide N-glycosidase F mutant of claim 1.
3. The nucleotide molecule according to claim 2, characterized in that, The nucleotide sequence is shown in SEQ ID NO.
2.
4. A recombinant plasmid comprising the nucleotide molecule of claim 2.
5. A recombinant cell comprising the nucleotide molecule of claim 2 or the recombinant plasmid of claim 4.
6. The use of the peptide N-glycosidase F mutant of claim 1, the nucleotide molecule of claim 2 or 3, the recombinant plasmid of claim 4, or the recombinant cell of claim 5 in deglycosylation or the preparation of deglycosylated products; The deglycosylation refers to the removal of N-linked oligosaccharides from glycoproteins.
7. The application according to claim 6, characterized in that, The application is the use of deglycosylation reactions in a standard or fast buffer system. The standard buffer system uses Glycoprotein Denaturing Buffer, GlycoBuffer 2, and NP-40 as buffer solutions. The rapid version of the buffer system uses Rapid PNGase F Buffer as the buffer solution.
8. A deglycosylation reagent, characterized in that, The reagent comprises the peptide N-glycosidase F mutant as described in claim 1.
9. A deglycosylation kit, characterized in that, The kit contains the peptide N-glycosidase F mutant as described in claim 1.