System for biosynthesis and fixed-point insertion of acetylated lysine in cells and application
By biosynthesis and site-point insertion of acetylated lysine in cells, the problems of low efficiency and high cost of acetylation introduction in the prior art are solved, efficient and accurate acetylation modification is achieved, and acetylation recombination is significantly improved. Protein yield and research value.
Patent Information
- Application Number
- CN202411269399.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-11
- Publication Date
- 2024-12-13
AI Technical Summary
The existing acetylation introduction technology has problems of low efficiency and high cost, and it is difficult to accurately control the acetylation regulation of proteins in cells. It is difficult for traditional methods to simulate the real state of acetylation, and the yield of acetylated recombinant proteins is low.
Provides a system for biosynthesis and site-directed insertion of acetylated lysines in cells, including genes encoding acetylated lysyl-tRNA synthetase and acetylated lysine synthetase, which is introduced into cells by genetic engineering, The biosynthesis and site-directed insertion of acetylated lysine in E. coli or mammalian cells is achieved, eliminating the step of exogenous addition of acetylated lysine.
It significantly improves the yield and efficiency of acetylated recombinant proteins, reduces the expression cost, realizes efficient and accurate acetylation modification in cells, and enhances the potential application value of protein acetylation research.
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Figure CN119120511A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of non-natural amino acid insertion, and specifically to a system and application for biosynthesizing and site-specifically inserting acetylated lysine in cells. Background Art
[0002] Protein acetylation, as an important part of post-translational modification, is catalyzed by histone acetyltransferases (HATs) and lysine acetyltransferases (KATs), which precisely transfer the acetyl group of acetyl-coenzyme A (AcCoA) to lysine, and the process is regulated by the concentration of AcCoA. Initially, the acetylation of histones was widely revealed and quickly attracted attention in the field of transcriptional regulation. Acetylation is not limited to histones, but also widely affects non-histone proteins. For example, at key sites of p53, acetylation fine-tunes its stability and function, profoundly affecting cell fate. This highlights the key role of lysine acetylation in life processes and inspires scientists to explore the acetylation mechanism and its effects in depth.
[0003] Scientists are curious: Can acetylation serve as a "switch" to precisely regulate protein function? How does it trigger a variety of changes in cells, including activity, stability, interaction, and localization? Does acetylation at different lysine sites give proteins more diverse regulatory capabilities? However, exploring these issues faces huge challenges. Under physiological conditions, the acetylation regulatory network is complex and difficult to control precisely. Traditional methods are also difficult to fully simulate the true state of acetylation. Therefore, innovation in the expression technology of acetylated recombinant proteins has become the key. We need more precise and efficient tools to modify acetylated proteins, reveal their regulatory mechanisms, and provide support for disease treatment and drug development.
[0004] Gene codon expansion (GCE) technology has greatly expanded the possibilities of protein research by introducing non-natural amino acids (ncAAs) into proteins. The core of GCE is the development of aaRS / tRNA pairs that are different from the natural translation mechanism of the cell. These enzymes can accurately recognize and bind to ncAAs. In previous studies, scientists used the acetylated lysyl tRNA synthetase KacRS / tRNA pair based on Methanosarcinamazei to insert chemically synthesized Kac into the target protein to obtain acetylated recombinant proteins. Although the above method adds up to 5-20mM Kac from an exogenous source, the yield of acetylated recombinant proteins is usually low, and the technical process is relatively complex and the expression cost is high. Summary of the invention
[0005] Purpose of the invention: In view of the efficiency and cost problems existing in the existing acetylation introduction technology, the present invention provides a system and application for the biosynthesis and site-specific insertion of acetylated lysine in cells. The system can be used to implement a "one-pot" protein site-specific acetylation modification strategy. The system can achieve the biosynthesis and site-specific insertion of acetylated lysine (Kac) in Escherichia coli or mammalian cells, and provides a specific application method of the system.
[0006] Technical solution: To achieve the above purpose, the present invention adopts the following technical solution:
[0007] In a first aspect, the present invention provides a system for biosynthesizing and site-specifically inserting acetylated lysine in a cell, the system comprising a gene encoding an acetylated lysine-tRNA synthetase, and a gene encoding an acetylated lysine synthetase having an amino acid sequence as shown in any one of SEQ ID NOs: 1-10.
[0008] As a specific embodiment, the cell is a prokaryotic cell or a mammalian cell; preferably, the cell is an Escherichia coli cell or a human cell.
[0009] As a specific embodiment, the amino acid sequence of the acetylated lysyl-tRNA synthetase is shown in SEQ ID NO: 11. The acetylated lysyl-tRNA synthetase is based on the acetylated lysyl-tRNA synthetase KacRS from Methanosarcinamazei.
[0010] As a specific embodiment, the system further comprises a tRNA paired with the gene encoding the acetylated lysyl-tRNA synthetase, the nucleotide sequence of which is shown in SEQ ID NO:12.
[0011] In a second aspect, the present invention provides the use of the system for biosynthesis and site-specific insertion of acetylated lysine in cells.
[0012] As a specific embodiment, the cell is a prokaryotic cell or a mammalian cell; preferably, the cell is an Escherichia coli cell or a human cell; more preferably, the cell is Escherichia coli BL21 (DE3) or BL21-AI, or a human embryonic kidney cell HEK293T.
[0013] As a specific implementation scheme, the application includes: co-transforming or transfecting a recombinant plasmid containing a gene encoding acetylated lysyl-tRNA synthetase and a recombinant plasmid containing an acetylated lysine synthetase gene with a target protein plasmid into cells for culturing, or co-transforming or transfecting a recombinant plasmid containing a gene encoding acetylated lysyl-tRNA synthetase and an acetylated lysine synthetase gene with a target protein plasmid into cells for culturing to obtain a target protein containing acetylated lysine; preferably, the target protein is sfGFP.
[0014] In a third aspect, the present invention provides a method for biosynthesizing and site-specifically inserting acetylated lysine in a cell, the method comprising co-transforming or transfecting a recombinant plasmid comprising a gene encoding an acetylated lysine-tRNA synthetase and a gene encoding an acetylated lysine synthetase with an amino acid sequence as shown in any one of SEQ ID NOs: 1-10 with a target protein plasmid into a cell for culturing, or co-transforming or transfecting a recombinant plasmid comprising a gene encoding an acetylated lysine-tRNA synthetase and a gene encoding an acetylated lysine synthetase with an amino acid sequence as shown in any one of SEQ ID NOs: 1-10 with a target protein plasmid into a cell for culturing, to obtain a target protein containing acetylated lysine.
[0015] In a fourth aspect, the present invention provides an acetylated lysine synthetase, wherein the amino acid sequence of the acetylated lysine synthetase is shown in any one of SEQ ID NOs: 1-10.
[0016] In a fifth aspect, the present invention provides the gene of the above-mentioned acetylated lysine synthetase.
[0017] In a sixth aspect, the present invention provides the acetylated lysine synthetase and the application of the gene in biosynthesis and site-directed insertion of acetylated lysine in cells.
[0018] Compared with the need for exogenous addition of Kac in the prior art, the system provided by the present invention eliminates this step and significantly increases the yield of acetylated recombinant proteins. Taking NmGNAT as an example, under the same culture conditions and time, when Kac is biosynthesized by NmGNAT and sfGFP is inserted at a fixed point, the protein yield of sfGFP149Kac is as high as 2.58mg / L, close to the yield of wild-type sfGFP protein 3.45mg / L; and when 5mM Kac is added exogenously, the protein yield of sfGFP149Kac is only 0.564mg / L, which is much lower than the biosynthesis group. Therefore, the present invention not only enriches the types of acetylated lysine synthetases, but also innovatively proposes a system and method for efficiently synthesizing and inserting Kac directly in Escherichia coli and mammalian cells without exogenous addition of Kac, which has important scientific research value and application prospects.
[0019] Beneficial effects: Compared with the traditional acetylation-modified recombinant protein expression technology, the present invention significantly improves the method and no longer needs to add chemically synthesized Kac exogenously to the culture medium. On the contrary, the present invention utilizes the aforementioned 10 acetylated lysine synthetases and acetylated lysyl tRNA synthetase KacRS / tRNA pairs, and through an innovative "one-pot method", directly incorporates the biosynthesized Kac site-specifically into the target protein in living cells. This method not only eliminates the chemical synthesis and purification steps of Kac, but also greatly improves the efficiency and specificity of recombinant protein acetylation modification. In the application of Escherichia coli and mammalian cell systems, NmGNAT showed the highest Kac biosynthesis efficiency, highlighting its potential application value in the field of protein acetylation research. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 This is an example of the site-specific acetylation modification process of the protein of the present invention.
[0021] Figure 2 This is a graph showing the relative fluorescence detection results for comparing the efficiency of Kac biosynthesis by acetyl-lysine synthetase in the Escherichia coli system.
[0022] Figure 3 The results of the biosynthesis of Kac by NmGNAT for site-directed insertion and expression of acetylated modified proteins are shown in Figure 1. a is the result of SDS-PAGE and WB; b is the result of mass spectrometry for the molecular weight of sfGFP 149Kac protein; c is the result of mass spectrometry for the protease-hydrolyzed peptide fragment of sfGFP 149Kac.
[0023] Figure 4 This is a flow cytometry result diagram of detecting the efficiency of acetyl-lysine synthetase biosynthesis Kac in a mammalian cell system.
[0024] Figure 5 This is the fluorescence imaging result of sfGFP 149Kac produced by biosynthesizing Kac using CeSSAT and NmGNAT in a mammalian cell system. DETAILED DESCRIPTION
[0025] The present invention is further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The experimental methods used in the examples are conventional methods unless otherwise specified, and the materials and reagents used are all commercially available unless otherwise specified.
[0026] The amino acids mentioned in the present invention are abbreviated as follows according to the nomenclature of IUPAC-IUB:
[0027] Alanine (Ala, A); Arginine (Arg, R); Asparagine (Asn, N); Aspartic acid (Asp, D); Cysteine (Cys, C); Glutamic acid (Glu, E); Glutamine (Gln, Q); Glycine (Gly, G); Histidine (His, H); Isoleucine (Ile, I); Leucine (Leu, L); Lysine (Lys, K); Methionine (Met, M); Phenylalanine (Phe, F); Proline (Pro, P); Serine (Ser, S); Threonine (Thr, T); Tryptophan (Trp, W); Tyrosine (Tyr, Y); Valine (Val, V)
[0028] In the examples, some abbreviations are as follows: Cm refers to chloramphenicol, Amp refers to ampicillin, Kan refers to kanamycin, Ara refers to L-arabinose, IPTG refers to isopropyl β-D-thiogalactoside, and NAM refers to nicotinamide.
[0029] The present invention discovers 10 new acetylated lysine synthetase genes through gene mining technology. The corresponding 10 acetylated lysine synthetases are named BcGNAT, BfSSAT, DsGNAT, NmGNAT, MgGNAT, BgGNAT, HsSSAT, AbGNAT, PsGNAT, and SeGNAT, respectively, and their amino acid sequences are shown in SEQ ID NOs: 1-10 in sequence.
[0030] Example 1 Biosynthesis and site-specific insertion of acetylated lysine in Escherichia coli
[0031] Using a genetic engineering strategy, the chemically synthesized gene fragments encoding BcGNAT, BfSSAT, DsGNAT, NmGNAT, MgGNAT, BgGNAT, HsSSAT, AbGNAT, PsGNAT and SeGNAT were connected to the pRSFDuet vector to obtain a vector plasmid expressing acetyl-lysine synthetase in Escherichia coli. By the same method, the gene fragments of the positive control enzyme CeSSAT (amino acid sequence as shown in SEQ ID NO: 13) and LYC1 (amino acid sequence as shown in SEQ ID NO: 14) were connected to the pRSFDuet vector to obtain a positive control plasmid in Escherichia coli. These 12 plasmids were co-transformed with the pBad-sfGFP-Asn149TAG (the protein expression gene contained therein is based on the wild-type sfGFP gene sequence, and the base sequence of the 149th amino acid of the sfGFP protein is mutated to TAG) plasmid into BL21 (DE3) or BL21-AI chemically competent cells containing the plasmid pEvol-MmKacRS (the vector contains the coding gene of MmKacRS with an amino acid sequence as shown in SEQ ID NO: 11, and a tRNA with a nucleotide sequence as shown in SEQ ID NO: 12), and the transformed cells were spread on LB solid culture medium containing 100 μg / mL Amp, 50 μg / mL Cm and 50 μg / mL Kan, and the plate containing the solid culture medium was placed upside down in a 37°C constant temperature incubator and cultured overnight. The 12 different monoclonal clones were added into 2 mL LB containing 100 μg / mL Amp, 50 μg / mL Cm and 50 μg / mL Kan according to the numbers 1 to 12, and cultured in a shaking incubator at 37°C until OD 600 When the OD value reached 0.6, the bacterial solution was evenly divided into two parts (negative control group and experimental group). A final concentration of 0.2% Ara, 0.2mMIPTG and 30mM NAM were added to the cell culture medium of the experimental group. No special treatment was given to the negative control group. After continuing to culture at 30°C for 16 hours, the cells were collected by centrifugation, the supernatant was removed, and an equal volume of PBS was added to resuspend the cells. The cells were resuspended and mixed. The absolute fluorescence value was divided by the OD value of each sample using Synergy H1 (BioTek, Winooski, VT, USA). 600 The relative fluorescence units (RFU) of the cells were determined by reading to compare the efficiency of the biosynthesis of Kac by the acetylated lysine synthetase. The fluorescence yield of wild-type sfGFP under the same conditions was used as a control. The OD value and fluorescence absorption value of the above samples were detected using an ELISA instrument. The results are as follows: Figure 2As shown. When the inducers Ara and IPTG were added to the culture medium, strong fluorescence values were detected in all twelve groups of samples; for the samples without the addition of inducers, no fluorescence values were detected, or the fluorescence values were low. Experiments have shown that in Escherichia coli, the ten newly identified acetylated lysine synthetases (i.e., BcGNAT, BfSSAT, DsGNAT, NmGNAT, MgGNAT, BgGNAT, HsSSAT, AbGNAT, PsGNAT, and SeGNAT) can all efficiently catalyze the production of Kac, and their efficiency is comparable to that of the positive controls CeSSAT and LYC1. Among them, the catalytic efficiency of the NmGNAT enzyme is the best and significantly better than the positive control. Using this strategy, we have successfully achieved the site-specific introduction of Kac in the target protein.
[0032] After the sfGFP-149Kac protein induced by NmGNAT was purified, its yield and purity reached the same level as that of the experimental group with exogenous addition of chemically synthesized Kac. The target bands can be clearly observed by SDS-PAGE and Western blot detection. Further analysis showed that the sfGFP-149Kac protein obtained by this method does contain acetylation modification, and its acetylation characteristic signal can be recognized by commercially available acetylation-specific antibodies. The specific results are as follows Figure 3 In addition, according to ESI-TOF mass spectrometry, the molecular weight of the sfGFP-149Kac protein purified by this method is completely consistent with the theoretical calculated value, as shown in Figure 3 b. Tandem MS mass spectrometry confirmed that the protein only contained acetylation modification at position 149, as shown in Figure 3 The above experimental results fully confirm that the method of the present invention can efficiently realize the biosynthesis of Kac in E. coli and accurately insert it into the designated position of the target protein without exogenous addition of Kac.
[0033] Example 2 Biosynthesis and site-specific insertion of acetylated lysine in mammalian cells
[0034] Using a genetic engineering strategy, gene fragments encoding 10 acetylated lysine synthetases, including BcGNAT, BfSSAT, DsGNAT, NmGNAT, MgGNAT, BgGNAT, HsSSAT, AbGNAT, PsGNAT and SeGNAT, as well as two expression genes CeSSAT (amino acid sequence as shown in SEQ ID NO: 13) and LYC1 (amino acid sequence as shown in SEQ ID NO: 14) as a positive control group, were connected to vectors
[0035] A plasmid was constructed to insert 12 acetylated lysine residues into pNEU-MmKacRS (the vector contains the gene encoding MmKacRS with the amino acid sequence shown in SEQ ID NO: 11 and the tRNA with the nucleotide sequence shown in SEQ ID NO: 12). 5 HEK293T cells were inoculated into 12-well plates and cultured for 24 hours. The cells were divided into negative control group, positive control group and experimental group. The negative control group was HEK293T cells without treatment. The 12 plasmids were co-transfected with pcDNA-sfGFP-Asn149TAG plasmid into HEK293T host cells, and a final concentration of 10mM NAM was added to the culture medium of the positive control group and experimental group cells. No treatment was performed in the negative control group. The cells were collected 48 hours after transfection; the cells were washed with 4°C PBS + 2% fetal bovine serum (FBS), centrifuged to collect the cells, the supernatant was removed, the cells were resuspended with 300μL PBS + 2% FBS and filtered with a 300-mesh flow filter cloth, and the fluorescence of the cells was measured by a MoFlo XDP flow cytometer using a 405 / 488nm laser (Beckmann Coulter, CA, USA). The results are shown in Figure 4 As shown in the figure, the fluorescence signal of the experimental group was significantly higher than that of the negative control group. The fluorescence signal of the NmGNAT enzyme was the strongest and significantly better than that of the positive control CeSSAT, indicating that its efficiency in catalyzing the production of Kac was optimal. The fluorescence imaging detection results showed that the efficiency of NmGNAT biosynthesis of Kac was equivalent to that of exogenously added 1mM Kac and that of the positive control group CeSSAT biosynthesis of Kac. The results are shown in the figure. Figure 5 The above experimental results prove that in mammalian cells, the aforementioned acetylated lysine synthetases can synthesize Kac in mammalian cells and insert it into the target protein at a specific site. Among them, NmGNAT has the highest efficiency in biosynthesis of Kac.
[0036] The above embodiments are the best implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.
Claims
1. A system for biosynthesis and site-specific insertion of acetylated lysine in cells, characterized in that: The system comprises a gene encoding an acetylated lysyl-tRNA synthetase and a gene encoding an acetylated lysine synthetase having an amino acid sequence as shown in any one of SEQ ID NOs: 1-10.
2. The system for biosynthesis and site-specific insertion of acetylated lysine in cells according to claim 1, characterized in that The cell is a prokaryotic cell or a mammalian cell; preferably, the cell is an Escherichia coli cell or a human cell.
3. The system for biosynthesis and site-specific insertion of acetylated lysine in cells according to claim 1, characterized in that The amino acid sequence of the acetylated lysyl-tRNA synthetase is shown in SEQ ID NO: 11; the system also includes a tRNA paired with the gene encoding the acetylated lysyl-tRNA synthetase, and its nucleotide sequence is shown in SEQ ID NO:
12.
4. Use of the system according to any one of claims 1 to 3 for biosynthesis and site-directed insertion of acetylated lysine in cells.
5. The use according to claim 4, characterized in that: The cell is a prokaryotic cell or a mammalian cell; preferably, the cell is an Escherichia coli cell or a human cell; more preferably, the cell is an Escherichia coli BL21 (DE3) or BL21-AI, or a human embryonic kidney cell HEK293T.
6. The use according to claim 4, characterized in that: The application includes: co-transforming or transfecting a recombinant plasmid containing a gene encoding acetylated lysyl-tRNA synthetase and a recombinant plasmid containing an acetylated lysine synthetase gene with a target protein plasmid into cells for culturing, or co-transforming or transfecting a recombinant plasmid containing a gene encoding acetylated lysyl-tRNA synthetase and an acetylated lysine synthetase gene with a target protein plasmid into cells for culturing, to obtain a target protein containing acetylated lysine; preferably, the target protein is sfGFP.
7. A method for biosynthesis and site-specific insertion of acetylated lysine in cells, characterized in that: The method comprises co-transforming or transfecting a recombinant plasmid containing a gene encoding an acetylated lysine-tRNA synthetase and a gene encoding an acetylated lysine synthetase with an amino acid sequence as shown in any one of SEQ ID NOs: 1-10 with a target protein plasmid into cells for culturing, or co-transforming or transfecting a recombinant plasmid containing a gene encoding an acetylated lysine-tRNA synthetase and a gene encoding an acetylated lysine synthetase with an amino acid sequence as shown in any one of SEQ ID NOs: 1-10 with a target protein plasmid into cells for culturing, thereby obtaining a target protein containing acetylated lysine.
8. An acetylated lysine synthetase, characterized in that The amino acid sequence of the acetylated lysine synthetase is shown in any one of SEQ ID NOs: 1-10.
9. A gene encoding the acetylated lysine synthetase according to claim 8.
10. Use of the acetylated lysine synthetase according to claim 8 and the gene according to claim 9 for biosynthesis and site-directed insertion of acetylated lysine in cells.