A method for regulating recombinant nuclease activity

Abstract

This invention discloses a method for regulating the activity of recombinant nucleases, belonging to the field of recombinant nuclease technology. The method involves cleaving the glycine-rich flexible linker peptide (Gly) in recombinant LbCas12a and LwCas13a nucleases using lysozyme (Lst). 4‑8 and (G4S) 1‑2 The method releases a fusion tag that influences nuclease activity, successfully enhancing the trans-cleavage activity of two nucleases. Lysocystis enzyme functions effectively over a wide temperature range (4–50°C) or alkaline conditions (pH 7.0–10.0), and is tolerant of certain concentrations of salt ions (20–500 mM). This method fills the gaps in current techniques, providing a new and convenient tool for regulating enzyme activity in recombinant nuclease research.

Description

Technical Field

[0001] This invention belongs to the field of recombinant nuclease technology, specifically relating to a method for regulating the activity of recombinant nucleases. Background Technology

[0002] CRISPR / Cas technology is a revolutionary gene editing and regulation tool that has rapidly become a cutting-edge research area in the biomedical field since its advent, and is widely used in gene regulation, disease treatment, nucleic acid detection, and bioimaging. The CRISPR / Cas system consists of clustered regularly spaced short palindromic repeats (CRISPR) and their associated nucleases (Cas). Cas nucleases mainly fall into two categories: Cas13a and Cas13b, which cleave RNA under RNA guidance, and Cas12a and Cas14, which cleave DNA under RNA guidance. Different Cas nucleases share a common characteristic: large molecular weight and low solubility during heterologous expression. They often require the fusion of solubilizing tags such as MBP-tag and SUMO-tag to improve their soluble recombination expression efficiency. However, the large molecular weight of MBP (40.3 kDa) and SUMO (11.2 kDa) tags can affect the correct folding of Cas nucleases, ultimately leading to reduced or even lost activity. Therefore, these solubilizing tags need to be removed downstream of Cas nuclease production to ensure the nuclease functions normally.

[0003] With oligoglycine (Gly) n and glycine-serine (G4S) n Flexible linker peptides, such as linkers, can maintain the relative flexibility or interaction between different linker domains in recombinant fusion enzymes, and are therefore often used to link nucleases to solubilizing tag domains. However, there are currently no literature reports on directly cleaving flexible linkers to remove solubilizing tags and regulate nuclease activity. Researchers can only achieve the cleavage target by introducing additional protease recognition sites, which increases the burden of preliminary design and gene manipulation, and the additional introduced sequences may interfere with the structure and expression characteristics of the nuclease. Therefore, there is a need to develop a convenient method for regulating nuclease activity to fill the gaps in current techniques and provide a new tool for enzyme activity regulation in recombinant nuclease research. Summary of the Invention

[0004] In order to overcome the shortcomings of the prior art, the present invention aims to provide a method for regulating the activity of recombinant nucleases, thereby solving the problems of the burden caused by the introduction of additional protease recognition sites and the possible interference with the structure and expression characteristics of nucleases.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] This invention discloses a method for regulating the activity of recombinant nucleases, which involves mixing and incubating a nuclease carrying a histidine tag, a solubilizing tag, and a glycine-rich flexible linker peptide with a lysozyme, and then using Ni... 2+ Affinity chromatography columns separate enzyme digestion mixtures. The eluting nickel column contains the tagged nuclease, while the adsorbed nickel column contains the cleaved tag, lysozyme, and uncleaved nuclease substrate.

[0007] Preferably, the nuclease includes recombinant LbCas12a and recombinant LwCas13a nucleases, the amino acid sequence of recombinant LbCas12a is shown in SEQ ID No. 1, and the amino acid sequence of recombinant LwCas13a nuclease is shown in SEQ ID No. 2.

[0008] Preferably, the amino acid sequence of the lysozyme is shown in SEQ ID No. 3.

[0009] Preferably, the lysozyme is produced by an Escherichia coli prokaryotic expression system and has a histidine tag at its C-terminus.

[0010] Preferably, the glycine-rich flexible linker peptide is (Gly). n Linker peptide or (G4S) m Linking peptides; wherein n is 4 to 8, the amino acid sequence of (Gly)4 is shown in SEQ ID No. 4, the amino acid sequence of (Gly)5 is shown in SEQ ID No. 5, the amino acid sequence of (Gly)6 is shown in SEQ ID No. 6, the amino acid sequence of (Gly)7 is shown in SEQ ID No. 7, the amino acid sequence of (Gly)8 is shown in SEQ ID No. 8, m is 1 or 2, the amino acid sequence of (G4S)1 is shown in SEQ ID No. 9, and the amino acid sequence of (G4S)2 is shown in SEQ ID No. 10.

[0011] Preferably, the molar ratio of nuclease to lysozyme is 0.1, 0.2, 0.4, 0.5, 0.8, 1.0, 1.5 or 2.0.

[0012] Preferably, the action temperature of lysozyme is 4–50°C.

[0013] Preferably, the pH value of the environment in which lysozyme acts is 7-10.

[0014] Preferably, lysozyme functions in the presence of sodium chloride at a final concentration of 20–500 mM.

[0015] Preferably, the Ni 2+ The affinity chromatography column is a nickel column.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention provides a method for regulating the activity of recombinant nucleases. The lysococcal enzyme used in this method can be produced by prokaryotic or eukaryotic expression systems such as Escherichia coli, Lactobacillus, Bacillus subtilis, Pichia pastoris, and goat mammary cells (the highest reported laboratory yield is 1315 mg / L). Therefore, compared with existing proteases that are expensive or difficult to produce, such as enterokinase, thrombin, and tobacco mosaic virus protease, lysococcal enzyme has significant advantages in terms of availability and application cost. This method successfully removed the 6*His-MBP tag from recombinant LbCas12a nuclease and the 6*His-SUMO tag from recombinant LwCas13a nuclease by cleaving the glycine-rich flexible linker peptide in recombinant nucleases with lysozyme. The removal of these tags significantly enhanced nuclease activity, achieving the goal of removing solubilizing tags and regulating nuclease activity. This avoids the operational burden and potential activity interference caused by introducing additional protease recognition sites, filling the gaps in current techniques, broadening the application scenarios of lysozyme, and enabling positive regulation of the activity of recombinant LbCas12a and LwCas13a nucleases. It provides a new tool for enzyme activity regulation in the field of recombinant nuclease technology. Furthermore, the technique of using lysozyme to cleave glycine-rich peptide chains to regulate protein function or enzyme activity can be extended to other research in the field of bioengineering, including structure-activity relationship studies, bioanalysis, and enzyme cross-linking reactions.

[0018] Furthermore, lysozyme can exert its regulatory effect over a wide temperature range (4℃~50℃), making it highly suitable for regulating the activity of temperature-sensitive nucleases. In addition, lysozyme exhibits similar activity under alkaline conditions ranging from pH 7.0 to 10.0, demonstrating good pH stability. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the method of the present invention for regulating recombinant nuclease activity by cleaving glycine-rich flexible linker peptides with lysozyme;

[0020] Figure 2These are gel images of the recombinant LbCas12a nuclease with oligoglycine linkers or glycine-serine linkers of different lengths before and after cleavage, according to the present invention. A is a gel image (SDS-PAGE, lane L is the protein standard marker) showing successful expression of the recombinant LbCas12a nuclease with oligoglycine linkers or glycine-serine linkers of different lengths; B is a gel image (SDS-PAGE, lane L is the protein standard marker) showing the effect of lysozyme cleavage of recombinant LbCas12a nuclease with oligoglycine linkers or glycine-serine linkers of different lengths.

[0021] Figure 3 This is a gel image showing the digestion of 6*His-MBP-8*Gly-LbCas12a recombinant nuclease by lysococcal enzyme and a comparative data graph of different LbCas12a nuclease activities. In this image, A is a gel image of 6*His-MBP-8*Gly-LbCas12a recombinant nuclease digested by lysococcal enzyme and LbCas12a recovered by nickel column affinity chromatography (SDS-PAGE, lane L is protein standard marker, lane #1 is 6*His-MBP-8*Gly-LbCas12a recombinant nuclease, lane #2 is lysococcal enzyme, lane #3 is the reaction mixture after incubation, lane U is the unbound sample during nickel column affinity chromatography, lane W is the rinse sample during nickel column affinity chromatography, and lane E is the eluted sample during nickel column affinity chromatography). B is a graph showing the activity data of different LbCas12a nucleases.

[0022] Figure 4 This image shows gel images of the 6*His-SUMO-8*Gly-LwCas13a recombinant nuclease digested by lysozyme of the present invention, as well as comparative data of different LwCas13a nuclease activities. A represents the gel images (SDS-PAGE) of 6*His-SUMO-8*Gly-LwCas13a recombinant nuclease digested by lysozyme and LwCas13a recovered by nickel column affinity chromatography. Lane L is the protein standard marker, lane #1 is the reaction mixture before incubation, lane #2 is the reaction mixture after incubation, lane U is the unbound sample during nickel column affinity chromatography, lane W is the rinse sample during nickel column affinity chromatography, and lane E is the eluted sample during nickel column affinity chromatography. B represents different LwCas13a nuclease activity data. Detailed Implementation

[0023] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0024] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0025] The present invention will now be described in further detail with reference to the accompanying drawings:

[0026] This invention provides a method for regulating the activity of recombinant nucleases, see [link to relevant documentation]. Figure 1 This includes the following steps:

[0027] Step 1: The recombinant nuclease carrying a histidine tag, a solubilizing tag, and a glycine-rich flexible linker peptide is mixed and incubated with lysostaphin (Lst). The lysostaphin removes the histidine tag and the solubilizing tag to regulate the activity of the recombinant nuclease.

[0028] The nucleases include recombinant LbCas12a and recombinant LwCas13a nucleases, whose corresponding amino acid sequences are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively. The amino acid sequence of the lysozyme is shown in SEQ ID No. 3. It can be produced using an E. coli prokaryotic expression system, has a histidine tag at the C-terminus, and can be purified to pure form using nickel column affinity chromatography, or it can be purchased directly (from Sigma-Aldrich). The glycine-rich flexible linker is oligoglycine (Gly). n Or glycine-serine (G4S) m Any of the linker peptides; wherein oligoglycine (Gly) is present. nThe linker peptide length n ranges from 4 to 8, and its corresponding amino acid sequence is shown in SEQ ID No. 4 to SEQ ID No. 8; glycine-serine (G4S) m The linker peptide length m is 1 or 2, and its corresponding amino acid sequence is shown in SEQ ID No. 9 and SEQ ID No. 10. The molar ratio of recombinant nuclease to lysin can be 0.1, 0.2, 0.4, 0.5, 0.8, 1.0, 1.5, or 2.0. The reaction temperature can be adjusted according to the characteristics of the recombinant nuclease, ranging from 4 to 50 °C. The pH value at which lysin functions is 7 to 10. The salt ion concentration that lysin tolerates is 20 to 500 mM. The relevant amino acid sequences are shown in Table 1.

[0029] Table 1 Amino acid sequence list

[0030]

[0031]

[0032]

[0033]

[0034] Step 2, using Ni 2+ Affinity chromatography (nickel column) separates enzyme digestion mixtures. The tagged nuclease flows directly out of the nickel column, while the tagged nuclease, lysozyme, and a very small amount of uncut nuclease substrate are adsorbed onto the nickel column, thereby achieving the separation and recovery of nuclease products.

[0035] The following detailed description of the method for regulating the activity of recombinant nucleases, with reference to specific embodiments, provides a concrete explanation.

[0036] 1. Expression and purification of recombinant LbCas12a nuclease with glycine-rich flexible linker peptide

[0037] Using the plasmid 6His-MBP-TEV-huLbCas12a (Addgene plasmid ID 90096) as a template, molecular cloning was used to replace the nucleotide sequences corresponding to the TEV protease recognition sites with nucleotide sequences corresponding to oligoglycine or glycine-serine linker peptides of different lengths. The successfully constructed and correctly sequenced vectors include 6His-MBP-3Gly-huLbCas12a, 6His-MBP-4Gly-huLbCas12a, 6His-MBP-5Gly-huLbCas12a, 6His-MBP-6Gly-huLbCas12a, 6His-MBP-7Gly-huLbCas12a, 6His-MBP-8Gly-huLbCas12a, 6His-MBP-(G4S)-huLbCas12a, and 6His-MBP-(G4S)2-huLbCas12a. The above eight vectors were transformed into *E. coli* expression strain BL21(DE3) to construct eight recombinant LbCas12 nuclease expression strains. Recombinant LbCas12a nuclease expression was induced for 16 hours at 20°C using IPTG at a final concentration of 0.5 mM. After expression, cells were disrupted using a freeze-thaw cycle combined with sonication, and Ni was used to... 2+ Eight recombinant nucleases were purified using affinity chromatography. The solution environment of the nucleases was replaced by phosphate buffer solution (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) by dialysis.

[0038] The purification of recombinant LbCas12a nucleases with oligoglycine or glycine-serine linker peptides of different lengths was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The results are as follows: Figure 2 As shown in Figure A, the electrophoretic bands of all eight samples were located between 150 and 250 kDa markers, which is consistent with the theoretical size of the recombinant LbCas12a nuclease (approximately 192 kDa), indicating that the eight recombinant nucleases were successfully expressed.

[0039] 2. Lysocysteine ​​cleavage of oligoglycine-linked peptides or glycine-serine-linked peptides of different lengths in recombinant LbCas12a nuclease.

[0040] The eight recombinant LbCas12a nucleases purified in Example 1 were mixed with lysozyme and incubated at 37°C for 3 hours. The lysozyme cleavage efficiency was detected using SDS-PAGE, and the results are as follows: Figure 2As shown in Figure B, lysozyme can recognize and cleave the flexible linker peptides in recombinant LbCas12a nuclease, including (Gly)4, (Gly)5, (Gly)6, (Gly)7, (Gly)8, (G4S), and (G4S)2. The sample after enzyme digestion showed an LbCas12a nuclease (without fusion tag) band at the 150kDa Marker in the gel image, which fully demonstrates the feasibility of lysozyme in removing the fusion tag.

[0041] The cleavage activity of lysozyme was measured using 6*His-MBP-8*Gly-LbCas12a recombinant nuclease and 6*His-SUMO-8*Gly-LwCas13a recombinant nuclease as examples.

[0042] 3. Determination of the trans-cleavage activity of lysozyme in 6*His-MBP-8*Gly-LbCas12a and different LbCas12a nucleases.

[0043] The purified 6*His-MBP-8*Gly-LbCas12a recombinant nuclease was mixed with lysozyme and incubated at 37°C for 3 hours. Ni 2+ Affinity chromatography (nickel column) was used to separate the mixture after lysin digestion: the tagged LbCas12a nuclease eluted directly from the nickel column, while the cleaved 6*His-MBP tag, lysin, and a very small amount of uncleaved recombinant nuclease substrate were adsorbed onto the nickel column. The recombinant nuclease was approximately 192 kDa before digestion; after digestion, the 6*His-MBP tag (approximately 43 kDa) was removed, yielding an LbCas12a nuclease with a theoretical size of approximately 144 kDa. Samples were taken at various key time points during the digestion and nickel column purification processes and analyzed using SDS-PAGE. The results are shown below. Figure 3 As shown in Figure A: In the sample incubated at 37°C (lane #3), a protein band appeared near the 150 kDa marker indicating the cleaved tag of LbCas12a, while a protein band appeared between the 37 and 50 kDa markers indicating the cleaved 6*His-MBP tag. The experimental results are as expected, indicating that lysozyme successfully cleaved the octane-glycine flexible linker peptide and removed the 6*His-MBP tag. Additionally, Ni... 2+ During the separation of the enzyme digestion mixture by affinity chromatography, a single band matching the size of LbCas12a nuclease appeared in the unbound sample (lane #U), while bands of 6*His-MBP tag and lysin appeared in the high-concentration imidazole eluted sample (lane #E), indicating that the affinity chromatography separation and recovery method is feasible. The trans-cleavage activity of 6*His-MBP-8*Gly-LbCas12a and the recovered LbCas12a nuclease after digestion was measured, and the results are as follows: Figure 3 As shown in Figure B, the initial cleavage rate of the fluorescent probe by LbCas12a was lower than that of the uncleaved recombinant nuclease, but ultimately more probes were cleaved (resulting in higher fluorescence values). In addition, the activity of LbCas12a recovered using this method was similar to that of commercial LbCas12a (NEB, catalog number M0653S), indicating that this method successfully achieved positive regulation of the activity of recombinant LbCas12a nuclease.

[0044] 4. Determination of the trans-cleavage activity of lysozyme in 6*His-SUMO-8*Gly-LwCas13a and different LwCas13a nucleases.

[0045] Using the pC013-Twinstrep-SUMO-huLwCas13a (Addgene plasmid ID 90097) plasmid as a template, a nucleotide sequence corresponding to the (Gly)8 flexible linker peptide was introduced between the nucleotide sequences corresponding to the SUMO and Cas13a groups via molecular cloning. The successfully constructed and correctly sequenced recombinant plasmid was named 6His-SUMO-8Gly-LwCas13a. The recombinant plasmid 6His-SUMO-8Gly-LwCas13a was transformed into the *E. coli* expression strain BL21(DE3) to construct a recombinant LwCas13a nuclease expression strain. Recombinant LwCas13a nuclease expression was induced for 16 hours at 20°C using IPTG at a final concentration of 0.5 mM. After expression, cells were disrupted using a freeze-thaw cycle combined with sonication, and Ni was used to... 2+ Recombinant LwCas13a nuclease was purified by affinity chromatography. The solution environment of the nuclease was replaced by phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) by dialysis.

[0046] The 6*His-SUMO-8*Gly-LwCas13a recombinant nuclease was mixed with an excess of lysozyme and incubated at 37°C for 3 hours. Ni 2+ Affinity chromatography was used to separate the mixture after lysin digestion: the tagged LwCas13a nuclease eluted directly from the nickel column, while the cleaved 6*His-SUMO tag, lysin, and a very small amount of uncleaved recombinant nuclease substrate were adsorbed onto the nickel column. The substrate size before digestion was approximately 156 kDa; after digestion, the 6*His-SUMO tag was removed, yielding the theoretically 139 kDa LwCas13a nuclease. Samples were taken at various key time points during the digestion and nickel column purification processes, and SDS-PAGE analysis was performed. The results are shown below. Figure 4As shown in Figure A. The gel image shows a significant downward shift in the position of the sample bands before and after incubation at 37°C (lanes #1 and #2), consistent with experimental expectations. This indicates that lysozyme successfully cleaved the octane-glycine flexible linker peptide and removed the 6*His-SUMO tag. Additionally, Ni... 2+ During the separation of the enzyme digestion mixture by affinity chromatography, a single band matching the size of LwCas13a nuclease appeared in the unbound sample (lane #U), and a band of lysin was visible in the high-concentration imidazole eluted sample (lane #E), indicating that the affinity chromatography separation and recovery method is feasible. The trans-cleavage activities of 6*His-SUMO-8*Gly-LwCas13a and the recovered LwCas13a nuclease were measured. Simultaneously, both nuclease samples were inactivated by treating them at 95℃ for 30 minutes. The inactivated samples served as negative controls for the activity test. The results are as follows: Figure 4 As shown in Figure B, LwCas13a outperforms the uncut recombinant nuclease in both initial cleavage rate and final number of probes cleaved. The calculated activity of LwCas13a nuclease is 1.19 times that of the uncut recombinant nuclease, indicating that this method successfully achieves positive regulation of the activity of recombinant LwCas13a nuclease.

[0047] The above experimental results show that lysozyme can successfully remove the 6*His-MBP tag from recombinant LbCas12a nuclease and the 6*His-SUMO tag from recombinant LwCas13a nuclease. The activity of the nucleases with the fusion tags removed is significantly improved. Therefore, this method can be effectively applied to the positive regulation of the activity of recombinant LbCas12a and LwCas13a nucleases.

[0048] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method of positively modulating the activity of a recombinant nuclease, comprising, The nuclease with a histidine tag, a solubility tag and a glycine-rich flexible linker peptide was mixed with lysostaphin and then incubated using a Ni 2+ The cleavage mixture was separated using a Ni affinity column. The flow-through from the column was the tagged nuclease, and the material that was adsorbed to the column was the cleaved tag, lysostaphin and uncut nuclease substrate. The nucleases are recombinant LbCas12a and recombinant LwCas13a nucleases, the amino acid sequence of the recombinant LbCas12a is shown as SEQ ID No. 1, the amino acid sequence of the recombinant LwCas13a nuclease is shown as SEQ ID No. 2, and the amino acid sequence of the lysostaphin is shown as SEQ ID No.

3.

2. The method for positively regulating recombinant nuclease activity according to claim 1, characterized in that, The lysostaphin is produced by an E. coli prokaryotic expression system, and the C-terminal has the histidine tag.

3. The method for positively regulating recombinant nuclease activity according to claim 1, characterized in that, The glycine-rich flexible linker is (Gly) n Linker or (G4S) m Linker, n is 4-8, and m is 1 or 2; The amino acid sequence of (Gly)4 is shown as SEQ ID No. 4, the amino acid sequence of (Gly)5 is shown as SEQ ID No. 5, the amino acid sequence of (Gly)6 is shown as SEQ ID No. 6, the amino acid sequence of (Gly)7 is shown as SEQ ID No. 7, the amino acid sequence of (Gly)8 is shown as SEQ ID No. 8, the amino acid sequence of (G4S)1 is shown as SEQ ID No. 9, and the amino acid sequence of (G4S)2 is shown as SEQ ID No.

10.

4. The method of claim 1-3, wherein the method comprises, The molar ratio of the nucleases to the lysostaphin is 0.1, 0.2, 0.4, 0.5, 0.8, 1.0, 1.5 or 2.

0.

5. The method of claim 1-3, wherein the method comprises, The lysostaphin has an action temperature of 4-50 ℃.

6. The method for positively regulating the activity of recombinant nuclease according to any one of claims 1 to 3, characterized in that, The lysostaphin has an action pH value of 7-10.

7. The method for positively regulating the activity of recombinant nuclease according to any one of claims 1 to 3, characterized in that, The lysostaphin has an action in the presence of sodium chloride at a final concentration of 20-500 mM.

8. The method for positively regulating the activity of recombinant nuclease according to any one of claims 1 to 3, characterized in that, The Ni 2+ The affinity chromatography column is a nickel column.

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