A mutant of cyclic gmp-amp synthetase and a method for preparing 2',3'-cyclic guanosine monophosphate-adenosine monophosphate using the same

Abstract

The application discloses a cyclic GMP-AMP synthetase mutant and a method for preparing 2',3'-cyclic guanosine monophosphate-adenosine monophosphate by using the cyclic GMP-AMP synthetase mutant, wherein the amino acid sequence of the cyclic GMP-AMP synthetase mutant has any one of the following mutations: P361A, L377M, K432R, S221Y / F357L / I496V, S221L / Y248F / K432H / S434T, F357L / P361A / L377M / S434T, F357L / P361A / S434T / H437T or H437T / S434T, compared with a wild type as shown in SEQ ID NO. 1. The activity of the cGAS mutant of the application can reach 4 times of the wild type, raw materials of the preparation method are economical and easy to obtain, the operation is simple, green, environment-friendly, pollution-free, the synthesis efficiency is high, and industrial development is easy.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a cyclic GMP-AMP synthase mutant and a method for preparing 2′,3′-cyclic guanosine monophosphate-adenosine monophosphate. Background Technology

[0002] 2′,3′-cyclic guanosine monophosphate-adenosine monophosphate (2′,3′-cyclic GMP-AMP, 2′,3′-cGAMP) is a naturally occurring cyclic dinucleotide second messenger molecule in living organisms, formed by the linkage of ATP and GTP, with the chemical formula C0. 20 H 24 N 10 O 13 P2, with a molecular weight of 674.41 and CAS number 1441190-66-4, exhibits a unique molecular structure. 2′,3′-cGAMP cyclizes guanosine monophosphate (GMP) with adenosine monophosphate (AMP) via unique 2′,5′ and 3′,5′ phosphodiester bonds. This distinctive structure enables it to precisely recognize and activate downstream interferon gene-stimulating factor (STING), a specific activation ability absent in other cyclic dinucleotides (such as 3′,3′-cGAMP and c-di-GMP). This structure underscores the irreplaceable role of 2′,3′-cGAMP in the regulation of innate immunity.

[0003] In the mammalian innate immune defense system, 2′,3′-cGAMP plays a core role as an "immune activation switch": when pathogens such as viruses and bacteria invade host cells, the double-stranded DNA (dsDNA) they carry is recognized and bound by cyclic guanosine monophosphate-adenosine monophosphate synthase (cyclic GMP-AMP synthase, cGAS enzyme) in the cytoplasm; the binding of dsDNA induces a conformational change in cGAS, transforming it from an inactive monomer into a catalytically active dimer, which in turn catalyzes the cyclization reaction of ATP and GTP to generate 2′,3′-cGAMP. The generated 2′,3′-cGAMP binds to the STING protein on the endoplasmic reticulum, prompting STING to translocate from the endoplasmic reticulum to the Golgi apparatus. Subsequently, it recruits and activates TBK1 kinase, which further phosphorylates the IRF3 transcription factor. After entering the nucleus, phosphorylated IRF3 initiates the expression of type I interferons (such as IFN-α / β) and a series of inflammatory factors (such as TNF-α and IL-6), ultimately establishing a broad-spectrum antiviral / antibacterial immune response.

[0004] In addition to its role in regulating innate immunity, 2′,3′-cGAMP also shows broad application prospects in the biomedical field: 1. 2′,3′-cGAMP can be used as an adjuvant for viral vaccines, enhancing antigen-specific T cell immune responses by activating the STING pathway, thus significantly improving vaccine efficacy. For example, adding 2′,3′-cGAMP adjuvant to the COVID-19 mRNA vaccine can increase the titer of neutralizing antibodies in mouse models by 3-5 times, and extend the duration of long-term immune memory to more than 6 months. 2. The solid tumor microenvironment contains a large number of "immunosuppressive cells" (such as regulatory T cells and tumor-associated macrophages), which prevents immune cells from effectively killing tumor cells. 2′,3′-cGAMP can activate dendritic cells (DCs) and cytotoxic T cells (CTLs) in the tumor microenvironment through local injection or intratumoral delivery, reversing the immunosuppressive state. Preclinical studies have shown that when 2′,3′-cGAMP is used in combination with PD-1 antibodies, the tumor inhibition rate against melanoma and lung cancer can reach over 70%, significantly superior to monotherapy. 3. For DNA virus infections such as herpes simplex virus (HSV) and cytomegalovirus (CMV), exogenous supplementation with 2′,3′-cGAMP can bypass the pathogen's inhibitory effect on cGAS (some viruses encode cGAS inhibitors, such as the ICP0 protein of HSV), directly activate the STING pathway, enhance the antiviral capacity of host cells, and reduce viral load.

[0005] In existing technologies, the commercial production of 2′,3′-cGAMP is mainly divided into chemical synthesis and enzyme catalysis. Chemical synthesis was the main method for preparing 2′,3′-cGAMP in the early stages. Its core idea is to construct the cyclized structure of 2′,3′-cGAMP through a three-step reaction of “protection-condensation-deprotection”. However, it has problems such as complicated reaction steps, difficulty in isomer separation and serious environmental pollution, which can lead to low yield, low product purity and poor safety. In comparison, enzyme catalysis is more environmentally friendly, with its core advantages being "one-step catalysis and green environmental protection." However, current technologies still face two major challenges: 1. Naturally derived cGAS (such as human, mouse, and bacterial cGAS) generally have low catalytic efficiency. Their active sites have weak affinity for ATP / GTP binding. When using wild-type human cGAS for catalysis, the conversion rate of 2′,3′-cGAMP is usually less than 12%, far from meeting the industrial production requirement of "conversion rate ≥50%". In addition, wild-type cGAS is sensitive to reaction conditions (such as temperature and pH), and is prone to denaturation and inactivation in environments above 37℃ or pH < 6.5. At the same time, the C-terminal regulatory domain of cGAS is easily degraded by proteases, leading to a rapid decline in enzyme activity. cGAS is usually used in whole-cell form and discarded after a single reaction, making it impossible to reuse and further increasing production costs. 2. In the enzyme-catalyzed reaction system, ATP and GTP raw materials are easily hydrolyzed, with a hydrolysis rate as high as 40%. A large amount of substrate is wasted, which not only increases production costs but also produces byproducts such as AMP and GMP, affecting subsequent purification.

[0006] Therefore, there is an urgent need in this field to develop a simple, easy-to-operate, economical, environmentally friendly, and substrate-utilization-efficient method for preparing 2′,3′-cyclic guanosine monophosphate-adenosine monophosphate (cGAS-ADP), which can solve the problems of low cGAS activity, severe substrate hydrolysis, and poor enzyme stability in existing methods, and achieve efficient synthesis of 2′,3′-cyclic guanosine monophosphate-adenosine monophosphate (cGAS-ADP) to adapt to the large-scale production of 2′,3′-cyclic guanosine monophosphate-adenosine monophosphate (cGAS-ADP), thus contributing to its industrialization research and development and application. Summary of the Invention

[0007] To address the above technical problems, in a first aspect, this application provides a cyclic GMP-AMP synthase mutant, wherein the amino acid sequence of the cyclic GMP-AMP synthase mutant, compared with the wild-type cyclic GMP-AMP synthase shown in SEQ ID NO.1, has any one of the following mutations:

[0008] P361A, L377M, K432R, S221Y / F357L / I496V, S221L / Y248F / K432H / S434T, F357L / P361A / L377M / S434T, F357L / P361A / S434T / H437T or H437T / S434T;

[0009] The amino acid sequences of the cyclic GMP-AMP synthase mutants with the S221L / Y248F / K432H / S434T mutation are shown in SEQ ID NO.2; the amino acid sequences of the cyclic GMP-AMP synthase mutants with the P361A mutation are shown in SEQ ID NO.3; the amino acid sequences of the cyclic GMP-AMP synthase mutants with the L377M mutation are shown in SEQ ID NO.4; the amino acid sequences of the cyclic GMP-AMP synthase mutants with the K432R mutation are shown in SEQ ID NO.5; the amino acid sequences of the cyclic GMP-AMP synthase mutants with the S221Y / F357L / I496V mutation are shown in SEQ ID NO.6; and the amino acid sequences of the cyclic GMP-AMP synthase mutants with the F357L / P361A / L377M / S434T mutation are shown in SEQ ID NO.4. As shown in ID NO.7, the amino acid sequence of the cyclic GMP-AMP synthase mutant with the F357L / P361A / S434T / H437T mutation is shown in SEQ ID NO.8, and the amino acid sequence of the cyclic GMP-AMP synthase mutant with the H437T / S434T mutation is shown in SEQ ID NO.9.

[0010] Specifically, the cyclic GMP-AMP synthase mutant further comprises one or more substitutions, additions, or deletions of amino acid residues, and has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.1.

[0011] Secondly, this application provides a nucleic acid molecule that encodes a cyclic GMP-AMP synthase mutant as described in the first aspect, or a complementary sequence to the encoding sequence.

[0012] Thirdly, this application provides a nucleic acid construct, which is an expression cassette. The nucleic acid construct contains the nucleic acid molecule described in the second aspect, and also contains a promoter, a transcription termination sequence, and a ribosome binding site.

[0013] Fourthly, this application provides a recombinant expression vector comprising the nucleic acid molecule described in the second aspect or the nucleic acid construct described in the third aspect, and the recombinant expression vector is a pET series vector suitable for expression in Escherichia coli.

[0014] Fifthly, this application provides a host cell containing the nucleic acid molecule described in the second aspect, the nucleic acid construct described in the third aspect, or the recombinant expression vector described in the fourth aspect, and the host cell is Escherichia coli BL21 (DE3).

[0015] In a sixth aspect, this application provides an enzyme preparation comprising the cyclic GMP-AMP synthase mutant described in the first aspect. The enzyme preparation is prepared by suspending whole-cell bacteria obtained by centrifuging the host cells described in the fifth aspect in a buffer solution, wherein the buffer solution is a Tris-HCl buffer or a potassium phosphate buffer, the pH of the buffer solution is 7-8, and the buffer solution contains 10%-20% glycerol.

[0016] In a seventh aspect, this application provides a kit comprising the enzyme preparation described in the sixth aspect, and further comprising ATP, GTP, CoCl2, double-stranded DNA, acetyl phosphate, and acetate kinase.

[0017] Eighthly, this application provides a method for preparing 2′,3′-cyclic guanosine monophosphate-adenosine monophosphate, the method comprising: using ATP and GTP as substrates, adding the aforementioned cyclic GMP-AMP synthase mutant as a catalyst to a buffer reaction system containing metal ions and double-stranded DNA, controlling the reaction temperature at 30-40°C, the pH of the reaction system at 5-10, and carrying out the enzyme-catalyzed reaction for 16-24 h to obtain 2′,3′-cyclic guanosine monophosphate-adenosine monophosphate;

[0018] In this process, the cyclic GMP-AMP synthase mutant participates in the reaction in the form of whole-cell bacteria obtained by centrifugation from host cells containing the cyclic GMP-AMP synthase mutant. The concentration of ATP is 2-20 mM, the molar ratio of ATP to GTP is 1:0.5 to 1:2, the mass of the catalyst is 50-300% of the mass of ATP, the metal ion is Co²⁺, Mn²⁺, Mg²⁺ or Zn²⁺, and the final concentration of the metal ion in the reaction system is 0.1-100 mM. The final concentration of the double-stranded DNA is 1-10 μM.

[0019] Preferably, the reaction temperature is 30°C, the pH of the reaction system is 6-9, and the enzyme-catalyzed reaction is carried out for 18-22 h.

[0020] More preferably, the pH of the reaction system is 7-8, and the enzyme-catalyzed reaction is carried out for 20 h.

[0021] Preferably, the concentration of ATP is 2-5 mM, the molar ratio of ATP to GTP is 1:1, the mass of the catalyst is 200% of the mass of ATP, the metal ion is Co²⁺, and the final concentration of the metal ion in the reaction system is 10-20 mM, and the final concentration of the double-stranded DNA is 1-2 μM.

[0022] Specifically, the reaction system also includes an ATP / GTP cycle system, which contains acetyl phosphate and acetate kinase. The molar ratio of acetyl phosphate to ATP in the reaction system is 1:1 to 1:3, and the mass ratio of acetate kinase to ATP in the reaction system is 1:3 to 1:6.

[0023] Preferably, the molar ratio of acetyl phosphate to ATP in the reaction system is 1:2, and the mass ratio of acetate kinase to ATP in the reaction system is 1:5.

[0024] Technical terms

[0025] The term "cGAS" refers to an enzyme that catalyzes the reaction of ATP and GTP to produce 2′,3′-cGAMP. Unless otherwise specified, the cGAS described in this application is a cyclic GMP-AMP synthase, also known as a cyclic guanosine monophosphate-adenosine monophosphate synthase. The wild-type cGAS used in this application is as follows: Homo sapiens (NP_612450.2, SEQ ID NO.1), Bacillus subtilis (WP_011197490.1) Homo sapiens (4KM5_A) Mus musclus (Q8C6L5.1) and Sus scrofa (I3LM39.1).

[0026] As used herein, the terms "cGAS mutant," "mutated cGAS," "mutant protein of cGAS," and "mutant protein of this application" are used interchangeably and all refer to cGAS that is not naturally occurring and includes cGAS that has been artificially modified based on the protein shown in SEQ ID NO: 1. Specifically, the cGAS mutant is as described in the first aspect of this application.

[0027] It should be understood that the amino acid numbers in the mutated cGAS of this application are all based on the wild-type cGAS (preferably, SEQ ID NO. 1). When a specific mutant protein has 80% or more homology with the sequence shown in SEQ ID NO. 1, the amino acid number of the mutant protein may be misaligned relative to the amino acid number in SEQ ID NO. 1, such as misalignment 1-5 positions towards the N-terminus or C-terminus of the amino acid. Using conventional sequence alignment techniques in the art, those skilled in the art can generally understand that such misalignment is within a reasonable range, and mutant proteins with the same or similar cGAS and 80% homology (e.g., 90%, 95%, 98%) should not be excluded from the scope of the cGAS mutants of this application due to the misalignment of amino acid numbers.

[0028] The cGAS mutant of this application can be a synthetic protein or a recombinant protein, that is, it can be a chemically synthesized product or produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, plants) using recombinant technology. Depending on the host used in the recombinant production protocol, the cGAS mutant of this application can be glycosylated or non-glycosylated. The cGAS mutant of this application may also include or exclude an initiating methionine residue.

[0029] This application also includes fragments, derivatives, and analogs of the cGAS mutant. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to proteins that substantially retain the same biological function or activity as the mutant protein.

[0030] The cGAS mutant fragments, derivatives, or analogs of this application may be (i) mutant proteins with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) substituted, such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) mutant proteins having substituent groups in one or more amino acid residues; or (iii) mutant proteins formed by fusing a mature mutant protein with another compound (such as a compound that extends the half-life of the mutant protein, for example, polyethylene glycol); or (iv) mutant proteins formed by fusing an additional amino acid sequence to the sequence of this mutant protein (such as a leader sequence or secretion sequence or a sequence used to purify this mutant protein or a proteogenic sequence, or a fusion protein formed with an antigen IgG fragment). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0031] Furthermore, the mutant protein of this application may also be modified. Modifications (generally without altering the primary structure) include chemically derived forms of the mutant protein, such as acetylation or carboxylation, either in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications performed during the synthesis and processing of the mutant protein or in further processing steps. This modification can be accomplished by exposing the mutant protein to glycosylating enzymes (such as mammalian glycosylation or deglycosylation enzymes). Modifications also include sequences containing phosphorylated amino acid residues (such as phosphotyrosine, phosphotyserine, phosphotythreonine). Modifications also include mutant proteins modified to improve their resistance to proteolytic hydrolysis or optimize their solubility.

[0032] This application also relates to nucleic acid molecules or polynucleotide sequences encoding the cGAS mutant of this application.

[0033] The polynucleotides of this application can be in the form of DNA or RNA. In one embodiment, the nucleotide is DNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to the sequence encoding the cGAS mutant of this application or a degenerate variant. As used herein, "degenerate variant" refers to a nucleic acid sequence encoding a polypeptide having the cGAS mutant of this application, but with a different coding region sequence.

[0034] This application also relates to variants of the aforementioned polynucleotides, which encode fragments, analogs, and derivatives of polypeptides or mutant proteins having the same amino acid sequence as those described herein. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the mutant protein it encodes. The cGAS and polynucleotides of this application are preferably provided in isolated form, and more preferably, purified to homogenate.

[0035] The nucleic acid sequence can be DNA, RNA, cDNA, or PNA. The nucleic acid sequence can be genomic, recombinant, or synthetic. The nucleic acid sequence can be isolated or purified. The nucleic acid sequence can be single-stranded or double-stranded. Preferably, the nucleic acid sequence will encode a cGAS mutant as described herein. The nucleic acid sequence can be derived by cloning, for example using standard molecular cloning techniques including restriction enzyme digestion, ligation, and gel electrophoresis, as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press). The nucleic acid sequence can be isolated, for example using PCR techniques. Isolation means separating the nucleic acid sequence from any impurities and from other nucleic acid sequences and / or proteins that are naturally found to associate with nucleic acid sequences in their source. Preferably, it will also be free of cell material, culture medium, or other chemicals from the purification / production process. The nucleic acid sequence can be synthetic, for example, produced by direct chemical synthesis. The nucleic acid sequence can be provided as naked nucleic acid or can be provided in combination with a protein or lipid.

[0036] The full-length nucleotide sequence or fragment thereof of the polypeptide of this application can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on publicly available nucleotide sequences, especially open reading frame sequences, and the relevant sequence can be amplified using a commercially available cDNA library or a cDNA library prepared according to conventional methods known to those skilled in the art. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified from each amplification are spliced ​​together in the correct order. Currently, it is possible to obtain the DNA sequence encoding the polypeptide (or its fragment, or its derivative) of this application entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.

[0037] This application also relates to vectors containing the polynucleotides of this application, and host cells genetically engineered using the vectors or polypeptide coding sequences of this application. The polynucleotides, vectors, or host cells described above can be isolated.

[0038] The host cell can be a prokaryotic cell, a lower eukaryotic cell, or a higher eukaryotic cell, such as mammalian cells (including human and non-human mammals).

[0039] DNA transformation of host cells can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *Escherichia coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0040] The obtained transformants can be cultured using conventional methods to express the protein encoded by the gene of this application. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture should be carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0041] The peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof.

[0042] The beneficial effects of this application include:

[0043] 1. The cyclic GMP-AMP synthase mutant provided in this application exhibits significantly enhanced activity compared to the wild type, with a conversion rate reaching up to 40.25%, which is four times that of the wild type (10.18% conversion rate), thus solving the core problem of low enzyme activity in the prior art. Furthermore, the cyclic GMP-AMP synthase mutant of this application has been optimized for multiple core sites, including the substrate binding domain, the double-stranded DNA binding domain, and the conformation of the active site, achieving a multi-dimensional improvement in catalytic efficiency.

[0044] 2. The method for preparing 2′,3′-cyclic guanosine monophosphate-adenosine monophosphate provided in this application, combined with the ATP / GTP cycling system of acetate kinase-acetyl phosphate, can increase the final conversion rate to 87.71%, improving the economic efficiency of the method. Compared with the existing creatine kinase-creatine phosphate system, the raw material cost of the cycling system in this application is reduced by 60%, and there is no metal ion competition between acetate kinase and cGAS, resulting in good compatibility of the method.

[0045] 3. The method for preparing 2′,3′-cyclic guanosine monophosphate-adenosine monophosphate provided in this application has mild and environmentally friendly reaction conditions, requires no organic reagents, meets the requirements of green production, and produces high-purity products without isomer impurities. Compared with chemical synthesis methods, the amount of waste generated by this application is significantly reduced, and production safety is significantly improved. In addition, this method has great industrialization potential. It uses whole cells of Escherichia coli as a catalyst, resulting in low preparation costs. The one-step catalytic reaction steps are simple and easy to operate, and standardized operations can be achieved through kits. The enzyme preparation is easy to store for a long time and can be reused 3-4 times, further reducing production costs. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of the enzyme-catalyzed reaction in Example 1.

[0047] Figure 2 The liquid chromatogram of the cGAS-A1 catalytic reaction to generate 2′,3′-cGAMP in Example 1 is shown.

[0048] Figure 3 The liquid chromatogram of the cGAS-B19 catalytic reaction in Example 2 to generate 2′,3′-cGAMP.

[0049] Figure 4 This is a schematic diagram of the enzyme-catalyzed reaction in Example 3.

[0050] Figure 5 The liquid chromatogram for the generation of 2′,3′-cGAMP using CoCl2 in Example 3 is shown.

[0051] Figure 6 This is a schematic diagram of the enzyme-catalyzed reaction in Example 4.

[0052] Figure 7 The liquid chromatogram for generating 2′,3′-cGAMP is shown in Example 4, which uses an enzymatic catalytic reaction based on an acetate kinase-acetyl phosphate ATP / GTP cycle. Detailed Implementation

[0053] The technical solutions of this application will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0054] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. All experimental materials used in this application are commercially available reagents unless otherwise specified. In the following examples, the cyclic GMP-AMP synthase mutant is abbreviated as cGAS, 2′,3′-cyclic guanosine monophosphate-adenosine monophosphate is abbreviated as 2′,3′-cGAMP, ATP is adenosine triphosphate, GTP is guanosine triphosphate, and dsDNA is double-stranded DNA (Cas No. 438545-06-3).

[0055] Example 1: Screening of wild-type cGAS

[0056] 1) Select cGAS wild type

[0057] Sequence analysis was performed on reported cGAS enzymes, and the enzymes ultimately selected from... Homo sapiens (genbank number NP_612450.2, SEQ ID NO.1), Bacillus subtilis (Genbank No. WP_011197490.1) Homo sapiens (Genbank No. 4KM5_A) Mus musclus (Genbank No. Q8C6L5.1) and Sus scrofa Further research will be conducted on cGAS (genbank No. I3LM39.1).

[0058] 2) Construction and transformation of recombinant vectors

[0059] 3-5 μL containing the target gene cGAS-A1 ( Homo sapiens The recombinant plasmid pET-30a-cGAS (synthesized by Genewiz) was added to 50-100 μL of E. coli BL21(DE3) competent cells. The cells were placed on ice for 20 min, subjected to heat shock at 42 °C for 90 sec, and then quickly returned to ice for 5 min. 800 μL of antibiotic-free LB medium was added, and the cells were cultured at 37 °C and 200 rpm for 1 h. The culture was then spread on LB agar plates containing kanamycin and cultured overnight at 37 °C for 12 h to obtain recombinant E. coli BL21(DE3) containing pET-30a-cGAS.

[0060] The formulation of antibiotic-free LB medium is as follows: yeast extract 5 g / L, sodium chloride 10 g / L, peptone 10 g / L;

[0061] The formulation of LB agar plate medium containing kanamycin resistance is as follows: yeast extract 5 g / L, sodium chloride 10 g / L, peptone 10 g / L, agar powder 20 g / L, kanamycin 50 μg / mL.

[0062] 3) Induced expression of expression vectors

[0063] The recombinant E. coli BL21(DE3) containing pET-30a-cGAS obtained in step 1) was directly plated onto a solid LB agar plate containing 50 μg / mL kanamycin resistance and cultured at 37 °C for 12–14 h to obtain single colonies. Single colonies of E. coli BL21(DE3) containing the pET-30a-cGAS recombinant vector were picked from the solid LB agar plate containing kanamycin and inoculated into 1 mL of liquid LB medium containing 50 μg / mL kanamycin resistance, and cultured at 37 °C with shaking for 12 h. Then, at a 5% (v / v) inoculation rate, they were separately inoculated into 1 L of fresh M9 medium containing kanamycin resistance and cultured at 37 °C until OD. 600 When the concentration was approximately 0.6-0.8, IPTG (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to a final concentration of 1.0 mM, and expression was induced at 200 rpm and 25 ℃ for 20 h.

[0064] The formulation of M9 medium with kanamycin resistance is as follows: K2HPO4 12.5 g / L, KH2PO4 6.25 g / L, yeast extract 6.67 g / L, (NH4)2SO4 1.76 g / L, glycerol 1 g / L, PPG2000 (antifoam) 0.2 g / L, MgSO4 0.26 g / L, CaCl2 0.023 g / L, kanamycin resistance 50 mg / L.

[0065] 4) Preparation of whole-cell bacterial cells

[0066] Centrifuge the bacterial culture from step 3) at 4°C, 4000 rpm for 30 min, remove the supernatant, and obtain whole-cell bacterial cells. Store the bacterial cells at -30°C.

[0067] Using the same steps as above, the remaining four different wild-type whole-cell bacterial cells were prepared. The whole-cell bacterial cells of all cGAS mutants mentioned in the subsequent examples were also prepared using the above method.

[0068] 5) Enzymatic reaction efficiency of cGAS from different sources

[0069] according to Figure 1The reaction was performed as shown: Add 5 mM of compound I ATP, 5 mM of compound II GTP, 15 mM MgCl2, 1.5 μM dsDNA (Cas No. 438545-06-3, purchased from SIGMA-ALDRICH), 150 mM NaCl, and 2 mL Tris-HCl buffer (pH 7.5, 200 mM) to a 4 mL reaction flask. After mixing thoroughly, add cGAS (whole-cell bacteria) at 200% of the ATP mass and incubate at 30°C and 1000 rpm for 20 hours. After the reaction, transfer 100 μL of the reaction solution to a 1.5 mL centrifuge tube, add 0.9 mL of 50% (v / v) methanol aqueous solution to terminate the reaction, centrifuge the tube at 14000 rpm for 1 min, and analyze the supernatant using UPLC.

[0070] The analytical conditions were as follows: the column was an Agilent Poroshell 120 Hilic-Z column; mobile phase A was 10 mM NH4OAc in water : ACN=95:5 (v / v), and mobile phase B was 10 mM NH4OAc in water : ACN=5:95 (v / v); the gradient elution program is shown in Table 1 below; the temperature was 30 ℃; the flow rate was 0.5 mL / min; and the detection wavelength was 254 nm.

[0071] Table 1

[0072]

[0073] The conversion rate of 2′,3′-cGAMP is calculated as the percentage of the product peak area (Area) in liquid chromatography. The conversion rate of 2′,3′-cGAMP, Conv(2′,3′-cGAMP) % = Area[2′,3′-cGAMP / (2′,3′-cGAMP + dA + Ade + dG + Gua + ATP + ADP + AMP + GTP + GDP + GMP)]*100%

[0074] The reaction results of cGAS from different sources are shown in Table 2. The liquid chromatogram of the cGAS-A1 catalytic reaction to generate 2′,3′-cGAMP is shown in Table 2. Figure 2 As shown.

[0075] Table 2

[0076]

[0077] Table 2 combined Figure 2It can be seen that cGAS-A1 (from Homo sapiens The catalytic efficiency of the wild-type genbank number NP_612450.2 (SEQ ID NO.1) is the highest, so a mutation was introduced based on this wild-type and subsequent studies were conducted.

[0078] Example 2 Enzymatic reaction efficiency of different cGAS mutants

[0079] Based on the screening results of Example 1, mutations were introduced at the mutation positions shown in Table 3 for the wild-type cGAS-A1 with the amino acid sequence shown in SEQ ID NO.1, resulting in various cGAS mutants as shown in Table 3. Using the same substrate as in Example 1, the whole-cell cGAS enzyme was replaced with the whole-cell cGAS mutants shown in Table 3 (the whole-cell preparation method was the same as in Example 1), and the same reaction operation as in Example 1 was performed for the enzymatic reaction. The conversion results are shown in Table 3. The liquid chromatogram of the cGAS-B19-catalyzed reaction to generate 2′,3′-cGAMP is shown in Table 3. Figure 3 As shown.

[0080] Table 3

[0081]

[0082] As shown in Table 3 above, the mutated cGAS-B19 (amino acid sequence shown in SEQ ID NO.2) exhibits the highest catalytic efficiency, with a conversion rate four times that of the wild-type cGAS-A1. Other cGAS mutants with similarly good catalytic efficiency include: cGAS-B5 (SEQ ID NO.3), cGAS-B9 (SEQ ID NO.4), cGAS-B11 (SEQ ID NO.5), cGAS-B18 (SEQ ID NO.6), cGAS-B22 (SEQ ID NO.7), cGAS-B23 (SEQ ID NO.8), and cGAS-B28 (SEQ ID NO.9).

[0083] Example 3 Optimization of Metal Ions

[0084] The effect of different metal ions on the conversion rate was studied, and Mg was selected as the metal ion. 2+ (MgCl2), Zn 2+ (MgCl2), Co 2+ (MgCl2) and Mn 2+ (MnCl2).

[0085] The reaction formula is as follows Figure 4As shown, the reaction procedure was as follows: the metal ion concentration and other experimental procedures were the same as in Example 1. Specifically, 5 mM of compound I ATP, 5 mM of compound II GTP, 15 mM of metal ions, 1.5 μM of dsDNA, 150 mM of NaCl and 2 mL of Tris-HCl buffer (pH 7.5, 200 mM) were added to a 4 mL reaction flask. After stirring evenly, cGAS-B19 (whole cells, amino acid sequence as shown in SEQ ID NO.2) with a mass of 200% of the ATP mass was added, and the mixture was placed in a shaker at 30°C and 1000 rpm for 20 hours.

[0086] After the reaction was complete, 100 μL of the reaction solution was added to a 1.5 mL centrifuge tube, and 0.9 mL of 50% (v / v) methanol aqueous solution was added to terminate the reaction. The centrifuge tube was centrifuged at 14000 rpm for 1 min, and the supernatant after centrifugation was analyzed by UPLC. The reaction results are shown in Table 4. The HPLC chromatogram of the 2′,3′-cGAMP generated by the enzymatic catalysis of CoCl2 is shown in Table 4. Figure 5 As shown.

[0087] Table 4

[0088]

[0089] Based on Table 4 Figure 5 It can be seen that using CoCl2 can improve the conversion rate of the enzymatic reaction, and the conversion rate is 1.6 times that of using MgCl2. Therefore, Co is the metal ion chosen. 2+ .

[0090] Example 4: Using the combined ATP / GTP cycling system to improve the conversion rate of enzymatic reactions

[0091] In the process of cGAS catalyzing the reaction of ATP and GTP to generate 2′,3′-cGAMP, spontaneous hydrolysis of ATP and GTP often occurs. ATP is hydrolyzed to adenosine diphosphate (ADP), which is further hydrolyzed to adenosine monophosphate (AMP), deoxyadenosine (dA), and adenine (Ade). GTP is hydrolyzed to guanosine diphosphate (GDP), which is further hydrolyzed to guanosine monophosphate (GMP), deoxyguanosine (dG), and guanine (Gua), leading to a decrease in conversion rate. Therefore, this application also discloses a preparation method that uses an ATP / GTP cycle system to improve the efficiency of enzymatic reactions, which can utilize the hydrolyzed ADP / GDP to generate ATP / GTP, thereby improving the conversion rate of enzymatic reactions.

[0092] The reaction formula is as follows Figure 6As shown, the reaction procedure was as follows: using the same substrate, based on the reaction procedure of Example 3 (metal ion CoCl2), the conversion rate was compared between the system with and without the addition of acetate kinase-acetyl phosphate ATP / GTP cycling. In the acetate kinase-acetyl phosphate ATP / GTP cycling system, the molar ratio of acetyl phosphate to ATP was 1:2, and acetate kinase (from...) Thermotoga maritima The mass ratio of 2′,3′-cGAMP (genbank ID AKE29949.1) to ATP was 1:5. The reaction results are shown in Table 5. The combined use of an acetate kinase-acetyl phosphate ATP / GTP cycle system to carry out the enzymatic catalytic reaction to generate 2′,3′-cGAMP is shown in the liquid chromatogram. Figure 7 As shown.

[0093] Table 5

[0094]

[0095] As shown in Table 5, the conversion rate was increased to 87.71% when the combined use of the acetate kinase-acetyl phosphate ATP / GTP cycle system with cGAS for enzymatic reaction.

[0096] In summary, the above embodiments are merely preferred embodiments of this application and are not intended to limit the scope of protection of this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A cyclic GMP-AMP synthase mutant, characterized in that, The amino acid sequence of the cyclic GMP-AMP synthase mutant is derived from the wild-type cyclic GMP-AMP synthase shown in SEQ ID NO.1 by mutation, wherein the mutation is any one of the following: P361A, F357L / P361A / L377M / S434T or F357L / P361A / S434T / H437T; The amino acid sequences of the P361A mutated cyclic GMP-AMP synthase mutants are shown in SEQ ID NO.3, the amino acid sequences of the F357L / P361A / L377M / S434T mutated cyclic GMP-AMP synthase mutants are shown in SEQ ID NO.7, and the amino acid sequences of the F357L / P361A / S434T / H437T mutated cyclic GMP-AMP synthase mutants are shown in SEQ ID NO.

8.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the cyclic GMP-AMP synthase mutant as described in claim 1, or is a complementary sequence to the coding sequence.

3. A nucleic acid construct, characterized in that, The nucleic acid construct is an expression cassette, which contains the nucleic acid molecule as described in claim 2, and further contains a promoter, a transcription termination sequence, and a ribosome binding site.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule of claim 2 or the nucleic acid construct of claim 3, and the recombinant expression vector is a pET series vector suitable for expression in Escherichia coli.

5. A host cell, characterized in that, The host cell contains the nucleic acid molecule of claim 2, the nucleic acid construct of claim 3, or the recombinant expression vector of claim 4, and the host cell is Escherichia coli BL21(DE3).

6. An enzyme preparation, characterized in that, The enzyme preparation comprises the cyclic GMP-AMP synthase mutant of claim 1. The enzyme preparation is prepared by suspending whole cells obtained by centrifugation of the host cells of claim 5 in a buffer solution. The buffer solution is Tris-HCl buffer or potassium phosphate buffer, the pH of the buffer solution is 7-8, and the buffer solution contains 10%-20% glycerol.

7. A reagent kit, characterized in that, The kit contains the enzyme preparation of claim 6, and also contains ATP, GTP, CoCl2, double-stranded DNA, acetyl phosphate, and acetate kinase.

8. A method for preparing 2′,3′-cyclic guanosine monophosphate-adenosine monophosphate, characterized in that, The method includes: using ATP and GTP as substrates, adding the cyclic GMP-AMP synthase mutant of claim 1 as a catalyst to a buffer reaction system containing metal ions and double-stranded DNA, controlling the reaction temperature at 30-40℃, the pH of the reaction system at 5-10, and carrying out the enzyme-catalyzed reaction for 16-24h to obtain 2′,3′-cyclic guanosine monophosphate-adenosine monophosphate; In this process, the cyclic GMP-AMP synthase mutant participates in the reaction in the form of whole-cell bacteria obtained by centrifugation from host cells containing the cyclic GMP-AMP synthase mutant. The concentration of ATP is 2-20 mM, the molar ratio of ATP to GTP is 1:0.5 to 1:2, the mass of the catalyst is 50-300% of the mass of ATP, the metal ion is Co²⁺, Mn²⁺, Mg²⁺ or Zn²⁺, and the final concentration of the metal ion in the reaction system is 0.1-100 mM. The final concentration of the double-stranded DNA is 1-10 μM.

9. The method for preparing 2′,3′-cyclic guanosine monophosphate-adenosine monophosphate according to claim 8, characterized in that, The concentration of ATP is 2-5 mM, the molar ratio of ATP to GTP is 1:1, the mass of the catalyst is 200% of the mass of ATP, the metal ion is Co²⁺, and the final concentration of the metal ion in the reaction system is 10-20 mM, and the final concentration of the double-stranded DNA is 1-10 μM.

10. The method for preparing 2′,3′-cyclic guanosine monophosphate-adenosine monophosphate according to claim 8, characterized in that, The reaction system also includes an ATP / GTP cycle system, which contains acetyl phosphate and acetate kinase. The molar ratio of acetyl phosphate to ATP in the reaction system is 1:1 to 1:3, and the mass ratio of acetate kinase to ATP in the reaction system is 1:3 to 1:6.

Images