Modified firefly luciferase, preparation method therefor and use thereof

Abstract

Provided in the present invention are a series of substrate binding pocket-modified firefly luciferases having high enzyme activity and high thermal stability, a preparation method therefor and the use thereof. Through the analysis of the spatial structure of the substrate binding pocket of the firefly luciferase, and on the basis of considering a large number of amino acid sites related to enzyme activity and thermal stability of firefly luciferase, the present inventors design, construct and detect a series of firefly luciferase mutants. The firefly luciferase has a high enzyme activity and thermal stability.

Description

Modified firefly luciferase, preparation method and application thereof

[0001] This application claims priority to patent application No. CN 202310958770.6, filed on August 1, 2023; the entire contents of which are incorporated herein. Technical Field

[0002] The present invention belongs to the field of biotechnology; more specifically, the present invention relates to a modified firefly luciferase, a preparation method and application thereof; the firefly luciferase has high enzyme activity and thermal stability. Background Art

[0003] Firefly luciferase catalyzes the oxidation of firefly luciferin in an aerobic environment, accompanied by bioluminescence. This reaction requires magnesium ions and ATP. In the presence of excess firefly luciferase and luciferin, the intensity of the luminescence produced is proportional to the amount of ATP. Therefore, firefly luciferase is widely used in biotechnology for ATP measurement. In previous studies, the present inventors have shown that by introducing mutations into wild-type firefly luciferase, the enzymatic activity and thermal stability of luciferase can be improved.

[0004] With the advancement of technology, this field has put forward higher requirements for applications such as cell viability detection. In order to better apply this technology, it is urgent to further improve the enzymatic activity and thermal stability of firefly luciferase.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a series of firefly luciferase mutants with modified substrate binding pockets, and preparation methods and applications thereof; the firefly luciferase has high enzyme activity and thermal stability.

[0007] In the first aspect of the present invention, a method for improving the enzymatic activity and / or thermal stability of firefly luciferase is provided, comprising: mutating the firefly luciferase to form a firefly luciferase mutant, wherein the mutant is adjacent to the substrate binding pocket in spatial structure and contributes to the recognition and binding of the substrate or the stabilization of the protein's spatial conformation.

[0008] In another aspect of the present invention, a firefly luciferase substrate binding pocket-modified mutant is provided. The mutant is spatially adjacent to the substrate binding pocket and contributes to substrate recognition and binding or stabilization of the protein's spatial conformation. The mutant has an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 2, and undergoes the following mutations (including combined mutations):

[0009] A. A mutation selected from (a)-(t) or a combination thereof:

[0010] (a) amino acid F at position 293 mutated to L (M1);

[0011] (b) amino acid Y at position 254 mutated to S (M2);

[0012] (c) position 239 mutated from V to A (M5);

[0013] (d) position 137 mutated from N to K, position 235 mutated from A to T, position 280 mutated from K to R, position 333 mutated from N to D, and position 422 mutated from I to V (M13);

[0014] (e) position 57 mutated from K to R (M14);

[0015] (f) position 42 mutated from T to A, position 282 mutated from E to D, position 392 mutated from K to I, and position 426 mutated from D to V (M15);

[0016] (g) position 525 mutated from S to P (M17);

[0017] (h) amino acid position 231 is mutated from N to A, R, or K (M20, M21, or M22);

[0018] (i) amino acid A at position 229 is mutated to R or K (M23 or M24);

[0019] (j) position 229 mutated from A to K, and position 231 mutated from N to K (M25);

[0020] (k) position 231 mutated from N to K, and position 293 mutated from F to L (M27);

[0021] (l) position 229 mutated from A to R, and position 231 mutated from N to R (M29);

[0022] (m) position 231 mutated from N to R, and position 293 mutated from F to L (M30);

[0023] (n) position 229 mutated from A to K, and position 231 mutated from N to A (M31);

[0024] (o) position 231 mutated from N to A, and position 293 mutated from F to L (M33);

[0025] (p) position 229 mutated from A to K, position 231 mutated from N to K, and position 293 mutated from F to L (M34);

[0026] (q) position 229 mutated from A to R, position 231 mutated from N to K, and position 293 mutated from F to L (M35);

[0027] (r) position 229 mutated from A to K, position 231 mutated from N to R, and position 293 mutated from F to L (M36);

[0028] (s) position 229 mutated from A to R, position 231 mutated from N to R, and position 293 mutated from F to L (M37);

[0029] (t) position 229 mutated from A to K, position 231 mutated from N to K, and position 293 mutated from F to L (M38);

[0030] B. A protein derived from protein A, which is formed by substituting, deleting, or adding one or more (e.g., 1-20; preferably 1-15; more preferably 1-10, such as 5 or 3) amino acid residues in the amino acid sequence of protein A and has the function of protein A, but the positions corresponding to (a)-(t) in A are conserved (the amino acid sequence does not change);

[0031] C. A protein derived from A that has more than 80% (preferably more than 85%; more preferably more than 90%; more preferably more than 95%, such as 98%, 99%) homology with the amino acid sequence of A protein and has the function of A protein, but the sites corresponding to (a)-(t) in A are conserved (the amino acid sequence does not change).

[0032] In one or more preferred embodiments, the stability includes: thermal stability.

[0033] In one or more preferred embodiments, the temperature stability or thermal stability includes temperature stability or thermal stability at a higher temperature of 37°C or above, preferably 40°C or above, more preferably 42°C or above, more preferably 45°C or above, more preferably 48°C or above, more preferably 50°C or above.

[0034] In one or more preferred embodiments, the mutated firefly luciferase includes the sequence segment in SEQ ID NO: 2 that does not carry a signal peptide.

[0035] In another aspect of the present invention, a polynucleotide is provided, wherein the polynucleotide encodes the firefly luciferase mutant.

[0036] In another aspect of the present invention, a vector or a genetically engineered host cell containing the vector is provided, wherein the vector contains the polynucleotide; or the host cell contains the vector or the polynucleotide is integrated into its genome.

[0037] In one or more preferred embodiments, the host cells include: prokaryotic cells or eukaryotic cells; preferably, the eukaryotic host cells include: yeast cells, fungal cells, insect cells, mammalian cells, etc.; the prokaryotic host cells include Escherichia coli, Bacillus subtilis, etc.

[0038] In one or more preferred embodiments, the host cell is a yeast cell.

[0039] In one or more preferred embodiments, the vector further comprises a signal peptide, a tag polypeptide, a fluorescent protein, and / or a promoter at the 5' end of the polynucleotide, and further comprises a tag polypeptide, a fluorescent protein, and / or a terminator at the 3' end of the polynucleotide.

[0040] In one or more preferred embodiments, compared with the firefly luciferase with the amino acid sequence shown in SEQ ID NO: 2, the enzyme activity of the modified firefly luciferase is increased by more than 20%; preferably increased by more than 50%; more preferably increased by more than 100%; and more preferably increased by more than 150%.

[0041] In one or more preferred embodiments, compared with the firefly luciferase with the amino acid sequence shown in SEQ ID NO: 2, the thermal stability of the modified firefly luciferase is significantly improved by more than 1.6 times, preferably more than 2.5 times, more preferably more than 2.6 times, more preferably more than 2.7 times, such as 2.8 times.

[0042] In another aspect of the present invention, a method for producing the firefly luciferase mutant is provided, comprising the steps of: (i) culturing the host cell; (ii) collecting the culture containing the firefly luciferase mutant; and (iii) isolating the firefly luciferase mutant from the culture.

[0043] In another aspect of the present invention, the use of the firefly luciferase mutant, the host cell expressing the mutant, or its lysate is provided for catalyzing the oxidation of firefly luciferase substrates, generating ATP-dependent bioluminescence, and detecting cell number or cell viability.

[0044] In another aspect of the present invention, a method for catalytically oxidizing firefly luciferin to produce bioluminescence is provided, comprising: utilizing the firefly luciferase mutant, a host cell expressing the mutant, or a lysis product thereof to perform catalytic oxidation and produce bioluminescence in the presence of ATP.

[0045] In one or more embodiments, the catalysis is carried out in a system suitable for the reaction, which, for example, includes: metal ions (such as divalent metal ions) and oxygen; specifically, the metal ions include, for example: magnesium ions, calcium ions, zinc ions and / or manganese ions.

[0046] In one or more embodiments, the system suitable for the reaction further includes reagents selected from the group consisting of reaction buffer, sodium chloride, detergent, Gelatin, MES, quaternary ammonium salts such as CTAB, NaF, chelating agents such as EDTA, defoaming agents such as DF204, thiol-containing compounds such as DTT, phosphates, sulfites and thiosulfates, BSA other than Gelatin, glycerol and other protein stabilizers.

[0047] In one or more embodiments, the use or method is for in vitro ATP detection; preferably, the amount of ATP contained in a solution system is determined using the firefly luciferase mutant and luciferase substrate.

[0048] In one or more embodiments, the use or method is used for cell viability detection; preferably, the firefly luciferase mutant and luciferase substrate are mixed with cell culture to determine cell viability.

[0049] In one or more embodiments, the use or method is used for detecting cell number; preferably, the firefly luciferase mutant and luciferase substrate are mixed with cell culture to determine the cell number.

[0050] In another aspect of the present invention, a detection system or detection kit for catalyzing the oxidation of firefly luciferin and generating bioluminescence is provided, which contains the firefly luciferase mutant, or the host cell or its culture or lysate.

[0051] In one or more embodiments, the detection system or detection kit further includes firefly luciferin.

[0052] In one or more embodiments, the detection system further includes metal ions (such as divalent metal ions); preferably, the metal ions include magnesium ions, calcium ions, zinc ions and / or manganese ions.

[0053] In one or more embodiments, the detection system further includes reagents selected from the group consisting of reaction buffer, sodium chloride, detergent, Gelatin, MES, quaternary ammonium salts, chelating agents, defoaming agents, thiol-containing compounds, phosphates, sulfites, thiosulfates, BSA other than Gelatin, glycerol and other protein stabilizers.

[0054] In one or more embodiments, the detection system further includes a cell lysis reagent.

[0055] In one or more embodiments, the detection system further includes an ATP extraction reagent.

[0056] In another aspect of the present invention, an ATP tester is provided, comprising: a container containing the firefly luciferase mutant in the container; a container containing the luciferase substrate in the container; a sampling, adding and reaction device for adding a sample to be tested, wherein the sample to be tested is a sample that needs to be tested for ATP determination; preferably, a gas supply device is also included, and the gas contains oxygen.

[0057] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1. Comparison of the predicted three-dimensional structure and overall structure of firefly (Photuris pensylvanica) luciferase.

[0059] Figure 2. Three-dimensional structure analysis of firefly (Photuris pensylvanica) luciferase and display of the substrate binding pocket.

[0060] Figure 3. Comparison of enzyme activity detection of wild-type, 146-1H2, and mutant crude enzymes.

[0061] Figure 4 shows the electrophoresis patterns of firefly luciferases with significant effects in crude enzyme activity detection, as well as WT and 146-1H2 after expression and purification.

[0062] Figure 5. Comparison of enzyme activity detection of purified firefly luciferase.

[0063] Figure 6. Comparison of signal thermal stability detection of purified firefly luciferase.

[0064] Figure 7. Comparison of purified 146-1H2, M1, and M2 used for detection of different ATP concentrations.

[0065] Figure 8. Comparison of signal thermal stability of purified 146-1H2, M1, and M2 when used for detection of different ATP concentrations.

[0066] Figure 9. Comparison of purified M1 and M2 used for enzyme activity detection in different numbers of Hela cells. DETAILED DESCRIPTION

[0067] Firefly luciferase is a 545-aa protein, and the sites critically involved in its enzymatic activity and thermostability remain largely unexplored. Preliminary work on structural analysis and mutational engineering considered numerous amino acid sites, designing, constructing, and screening a series of mutants related to the substrate-binding pocket of firefly luciferase. After initial screening, 23 mutants with high enzymatic activity were expressed, purified, assayed, and tested for thermostability. Compared to the control (146-1H2), these mutants exhibited varying degrees of enhanced enzymatic activity and demonstrated excellent thermostability. Two mutants, M1 (F293L) and M2 (Y254S), exhibited significantly enhanced enzymatic activity, increasing by 182% and 172%, respectively, compared to the control (146-1H2). Furthermore, incubation at 50°C for 24 hours did not affect the enzymatic activity of these mutants, indicating their high thermostability.

[0068] the term

[0069] As used herein, unless otherwise specified, the "firefly luciferase mutant", "mutant firefly luciferase", "luciferase mutant", and "mutant luciferase" are used interchangeably and refer to an enzyme (polypeptide / protein) corresponding to the firefly luciferase before mutation, which is constructed after mutations occur at some sites determined by the inventors to be correlated with the enzyme activity / thermostability of the enzyme, preferably corresponding to the amino acid sequence shown in SEQ ID NO: 2, and the mutation is selected from the following sites or a combination thereof: position 293, position 254, position 229 or position 231, for a total of 23 firefly luciferase mutants.

[0070] If it is necessary to represent the firefly luciferase (146-1H2) before mutation, it can be an enzyme with an amino acid sequence such as SEQ ID NO: 2. Unless otherwise specified, the mutation sites of the mutants of the present invention are based on the sequence shown in SEQ ID NO: 2.

[0071] In the present invention, unless otherwise specified, the firefly luciferase mutant is identified by "amino acid replaced by the original amino acid position" to indicate the mutated amino acid in the firefly luciferase mutant, such as F293L, which means that the amino acid at position 293 is replaced by L from F in the starting enzyme.

[0072] As used herein, "isolated firefly luciferase" refers to a firefly luciferase mutant that is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Firefly luciferase mutants can be purified by one skilled in the art using standard protein purification techniques. Substantially pure protein will produce a single major band on a non-reducing polyacrylamide gel.

[0073] As used herein, "increased enzyme activity or signal thermostability" refers to a statistically significant increase, or a significant improvement, in the enzyme activity or signal thermostability of the mutated firefly luciferase compared to the original firefly luciferase polypeptide before modification. For example, under the same reaction conditions / environment, after a certain reaction time, the enzyme activity or signal thermostability of the mutant firefly luciferase with improved enzyme activity or signal thermostability is significantly increased by 5% or more, 10% or more, 20% or more, 30% or more, 50% or more, 70% or more, 80% or more, 100% or more, 150% or more, etc., compared to the original enzyme after modification.

[0074] As used herein, a "library of firefly luciferase mutants or polynucleotides encoding the same" refers to a collection of polypeptides or polynucleotides comprising a series of firefly luciferase mutants provided herein. The collection of multiple firefly luciferase mutants or polynucleotides encoding the same with varying enzymatic activity or signal thermostability into a single library facilitates those skilled in the art in selecting the appropriate firefly luciferase or its encoding nucleic acid based on desired reaction conditions.

[0075] As used herein, the terms "comprising" or "including" encompass "including," "consisting essentially of," and "consisting of." The term "consisting essentially of" means that the composition / reaction system / kit, in addition to the essential ingredients or components, may also contain minor components and / or impurities that do not affect the active ingredients in a small amount.

[0076] As used herein, the term "effective amount" refers to an amount that can produce a function or enzyme activity in the reaction of interest of the present invention and achieve the desired effect (accurate test results).

[0077] Firefly luciferase mutants, their encoding nucleic acids and constructs

[0078] The inventors used protein spatial structure prediction tools to predict the three-dimensional structure of firefly (Photuris pensylvanica) luciferase, and compared and analyzed the complex structure of firefly luciferase and substrate binding published in the PDB protein structure database (Protein Data Bank, PDB), confirming the uniqueness of the firefly luciferase mutant of the present invention.

[0079] The firefly luciferase mutants of the present invention can be products of chemical synthesis or produced using recombinant technology from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells).

[0080] The present invention also includes fragments, derivatives, and analogs of the firefly luciferase mutant. As used herein, the terms "fragment," "derivative," and "analog" refer to proteins that substantially retain the same biological function or enzymatic activity as the native firefly luciferase mutant of the present invention. The protein fragments, derivatives, or analogs of the present invention can be (i) proteins having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) proteins having a substituent group in one or more amino acid residues, or (iii) proteins formed by fusion of an additional amino acid sequence to the protein sequence (such as a leader sequence or secretory sequence, or a sequence used to purify the protein, a proprotein sequence, or a fusion protein). According to the definitions herein, these fragments, derivatives, and analogs fall within the scope known to those skilled in the art. However, the condition that must be met is that the amino acid sequence of the firefly luciferase mutant and its fragments, derivatives, and analogs must contain at least one of the mutations specifically described above in the present invention.

[0081] In the present invention, the term "firefly luciferase mutant" also includes (but is not limited to): deletions, insertions, and / or substitutions of several amino acids (usually 1-20, preferably 1-10, and even more preferably 1-8, 1-5, 1-3, or 1-2), as well as additions or deletions of one or several amino acids (usually within 20, preferably within 10, and more preferably within 5) at the C-terminus and / or N-terminus. For example, in the art, substitutions with amino acids having similar or similar properties generally do not alter protein function. For another example, additions or deletions of one or several amino acids at the C-terminus and / or N-terminus generally do not alter protein function. The term also includes enzymatically active fragments and enzymatically active derivatives of firefly luciferase mutants. However, these variants must contain at least one mutation described above in the present invention.

[0082] In the present invention, the term "firefly luciferase mutant" also includes (but is not limited to): derivative proteins that retain protease activity and share an amino acid sequence identity of 80% or greater, preferably 85% or greater, more preferably 90% or greater, and even more preferably 95% or greater, such as 98% or greater, or 99% or greater, with the firefly luciferase mutant. Similarly, these derivative proteins must contain at least one of the mutations described above.

[0083] The invention also provides analogs of the firefly luciferase mutant. The differences between these analogs and the firefly luciferase mutant may be differences in amino acid sequence, differences in modifications that do not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, site-directed mutagenesis or other known molecular biology techniques. Analogs also include analogs with residues other than natural L-amino acids (such as D-amino acids), as well as analogs with non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides exemplified above. Modifications (usually without changing the primary structure) include: chemical derivatization of the polypeptide in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modifications during polypeptide synthesis and processing or in further processing steps. Such modifications can be accomplished by exposing the polypeptide to a glycosylation enzyme (e.g., a mammalian glycosylase or deglycosylase). Modifications also include sequences containing phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides modified to increase their resistance to proteolysis or optimize their solubility.

[0084] The present invention also provides a polynucleotide sequence encoding the firefly luciferase mutant or its conservative variant protein of the present invention.

[0085] The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0086] The polynucleotide encoding the mature protein of the mutant includes: a coding sequence encoding only the mature protein; a coding sequence of the mature protein and various additional coding sequences; a coding sequence of the mature protein (and optional additional coding sequences) and non-coding sequences.

[0087] A "polynucleotide encoding a protein" may include a polynucleotide encoding the protein, or may include additional coding and / or non-coding sequences.

[0088] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the firefly luciferase mutant coding sequence, and a method for producing the mutant enzyme of the present invention by recombinant technology.

[0089] The polynucleotide sequences of the present invention can be used to express or produce recombinant firefly luciferase mutants using conventional recombinant DNA techniques. Generally, the following steps are involved:

[0090] (1) Transforming or transducing a suitable host cell with a polynucleotide encoding a firefly luciferase mutant (or variant) of the present invention, or a recombinant expression vector containing the polynucleotide;

[0091] (2) Host cells cultured in a suitable culture medium;

[0092] (3) Isolate and purify proteins from culture medium or cells.

[0093] In the present invention, the polynucleotide sequence of the firefly luciferase mutant can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well known in the art. In short, any plasmid or vector can be used as long as it can replicate and be stable in the host. A key feature of an expression vector is that it typically contains an origin of replication, a promoter, a marker gene, and translation control elements.

[0094] Methods well known to those skilled in the art can be used to construct expression vectors containing firefly luciferase mutant encoding DNA sequences and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. The expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selecting transformed host cells.

[0095] A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell to enable it to express the protein.

[0096] In the present invention, the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a fungal cell or a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples include Escherichia coli, Bacillus subtilis, Streptomyces, and Agrobacterium; and eukaryotic cells such as yeast and plant cells. In a specific embodiment of the present invention, Escherichia coli is used as the host cell.

[0097] Those skilled in the art will readily understand how to select appropriate vectors, promoters, enhancers, and host cells. In a preferred embodiment of the present invention, the expression vector used is pET22b; and the microbial host cells transformed with the expression vector are all Escherichia coli.

[0098] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0099] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its 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 renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, 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 of these methods.

[0100] Applications of Firefly Luciferase Mutants

[0101] Under suitable reaction conditions, firefly luciferase can catalyze the oxidation of firefly luciferin, producing bioluminescence; that is, the firefly luciferase of the present invention can be used as a catalyst in the ATP bioluminescence assay. The ATP bioluminescence assay is a simplified biochemical method that uses the reaction of ATP with a luciferin-luciferase complex to determine the presence of ATP. This method can be used to measure the amount of ATP in vitro or in cells. The bioluminescence reaction requires ATP, luciferin, and firefly luciferase. During the reaction, luciferin is oxidized and emits fluorescence. The number of photons can be measured using an ATP fluorimeter, and the number of photons is proportional to the ATP content. When applied to cell viability assays, since the ATP content in each cell is constant, the ATP content in a sample is related to the number of cells in the sample. This method can measure the amount of ATP very quickly (e.g., in seconds). Using the firefly luciferase mutants of the present invention, detection can be achieved more efficiently and sensitively.

[0102] After obtaining the firefly luciferase mutant enzyme of the present invention, according to the teachings of the present invention, those skilled in the art can conveniently apply the enzyme to catalyze the oxidation of firefly luciferin and produce bioluminescence in the presence of ATP. In a preferred embodiment, the catalysis is carried out in a system suitable for the reaction, which includes: metal ions (such as divalent metal ions, specifically magnesium ions) and oxygen; preferably, the system suitable for the reaction also includes (but is not limited to) reagents selected from the group consisting of: reaction buffer, sodium chloride, detergent, gelatin, MES, quaternary ammonium salts (such as CTAB), NaF, chelating agents (such as EDTA), defoaming agents (such as DF204), thiol-containing compounds (such as DTT), phosphates, sulfites, thiosulfates, BSA (excluding gelatin), glycerol and other protein stabilizers.

[0103] The application range of the ATP bioluminescence method based on firefly luciferase is very wide. It can be applied to many fields such as food (such as microbial detection), pharmaceuticals, electronics and cosmetics. For example, the bioluminescence method is used to determine the bacterial contamination in meat products. The ATP bioluminescence method can also be used for the determination of lactic acid bacteria in dairy products, the total colony count in beer, the bacteriological determination of condiments and dehydrated vegetables, etc. The ATP bioluminescence method can also be used for cleanliness detection in food production environments; for example, the ATP bioluminescence method can be used to detect the cleanliness of food production lines and kitchens, refrigerators, food control tables, railway station food utensils, etc. The ATP bioluminescence method can also be used for cleanliness detection in medical environments; for example, for microbial detection of surgical instruments and medical consumables.

[0104] The method of the present invention can also be applied to animal and plant tissue samples or cell samples to detect ATP levels to study biological metabolic performance, etc. For example, in the field of botany involving transgenic research, the ATP content of plant callus can be measured to study the characteristics of cellular energy metabolism therein.

[0105] It should be understood that the firefly luciferase mutants of the present invention having higher enzyme activity and thermal stability are suitable for use in a variety of ATP detection methods known or under development in the art and have broad applicability.

[0106] In a specific embodiment of the present invention, a number of Pennsylvania firefly luciferase mutants were established based on Luc146-1H2, and corresponding prokaryotic expression plasmids were constructed. After sequencing confirmation, the mutant plasmids were transformed into competent E. coli cells simultaneously with wild-type (WT) and control (146-1H2) plasmids. After cultivation and induction, E. coli bacteria expressing firefly luciferase were obtained. The cells were lysed to release the firefly luciferase, and the enzyme activities of the different mutants were compared. 23 mutants with higher enzyme activity and the control were cultured and induced for expression, and the firefly luciferase protein was purified. The Luc146-1H2 enzyme activity and signal thermostability of the mutants and the control were compared.

[0107] The present invention uses a method that combines molecular theoretical design and functional screening to obtain multiple firefly luciferase mutants with higher enzyme activity, all of which can be well used for quantitative ATP detection. Among them, the enzyme activity of mutant M1 (F293L) is approximately 182% higher than that of the control firefly luciferase, and the enzyme activity of mutant M2 (Y254S) is approximately 172% higher than that of the control firefly luciferase. The purified mutants and the control were heated at 50°C for 24 hours, and the enzyme activities before and after heating were compared. It was found that treatment at 50°C for 24 hours did not significantly affect the enzyme activity of firefly luciferase. Further testing found that this series of luciferase mutants can also be used to detect cell number and enzyme activity.

[0108] Composition / kit of firefly luciferase mutants

[0109] The present invention also provides a composition comprising an effective amount of the firefly luciferase mutant of the present invention and other components required for detection. Such components include (but are not limited to) components selected from the group consisting of firefly luciferin (as a substrate), metal ions (e.g., divalent metal ions), reaction buffer, sodium chloride, detergents, gelatin, MES, quaternary ammonium salts (e.g., CTAB), NaF, chelating agents (e.g., EDTA), defoaming agents (e.g., DF204), thiol-containing compounds (e.g., DTT), phosphates, sulfites, thiosulfates, BSA (excluding gelatin), glycerol, and other protein stabilizers. Those skilled in the art can determine the effective amount of the firefly luciferase mutant in the composition based on the actual use of the composition. Other substances that further modulate the enzymatic activity of the firefly luciferase mutant of the present invention or promote the reaction process may also be added to the composition.

[0110] The present invention also provides a detection system comprising: a firefly luciferase mutant of the present invention; firefly luciferin (as a substrate); and preferably further comprising components selected from the group consisting of: metal ions (e.g., divalent metal ions), reaction buffer, sodium chloride, detergent, gelatin, MES, quaternary ammonium salts (e.g., CTAB), NaF, chelating agents (e.g., EDTA), defoaming agents (e.g., DF204), thiol-containing compounds (e.g., DTT), phosphates, sulfites, thiosulfates, BSA (excluding gelatin), and protein stabilizers such as glycerol. In this detection system, upon addition of a test sample, a reaction occurs within a very short time, and the test result is obtained.

[0111] To facilitate expanded or commercial applications, the present invention also provides a detection system or detection kit, comprising: a firefly luciferase mutant of the present invention, firefly luciferin (as a substrate; preferably further comprising components selected from the group consisting of metal ions (such as divalent metal ions), reaction buffer, sodium chloride, detergent, gelatin, MES, quaternary ammonium salts (such as CTAB), NaF, chelating agents (such as EDTA), defoaming agents (such as DF204), thiol-containing compounds (such as DTT), phosphates, sulfites, thiosulfates, BSA other than gelatin, glycerol and other protein stabilizers. The firefly luciferase mutant, luciferin, metal ions (such as divalent metal ions), reaction buffer, sodium chloride, detergent, gelatin, MES and other reagents are independently placed in different containers, or two or more of them are mixed in the same container.

[0112] As a preferred embodiment of the present invention, the detection kit may further include: a cell lysis reagent, and / or an ATP extraction reagent.

[0113] As a preferred embodiment of the present invention, the detection kit may further include instructions for use to guide people to use the kit of the present invention in a correct manner.

[0114] To facilitate scalable or commercial applications, the present invention further provides an ATP tester comprising: a container containing the firefly luciferase mutant; a container containing a luciferase substrate (e.g., luciferin); a sampling (e.g., swab), sample addition, and reaction device for adding a sample to be tested, wherein the sample to be tested is a sample requiring ATP determination; and a gas supply device, wherein the gas comprises oxygen. Alternatively, naturally occurring air (oxygen) may be substituted for the gas supply device.

[0115] As a preferred embodiment of the present invention, the ATP tester further comprises: a container, and contained in the container: metal ions (such as divalent metal ions) (solution), reaction buffer, sodium chloride, detergent, gelatin, MES, quaternary ammonium salts (such as CTAB), NaF, chelating agents (such as EDTA), defoaming agents (such as DF204), thiol-containing compounds (such as DTT), phosphates, sulfites, thiosulfates, BSA (excluding gelatin), glycerol and other protein stabilizers. In the ATP tester, the firefly luciferase mutant, luciferin, metal ions (such as divalent metal ions), reaction buffer, sodium chloride, detergent, gelatin, MES and other reagents are independently placed in different containers, or two or more of them are mixed in the same container. The ATP tester can be provided with some calculation programs and an output screen to intuitively display the measurement results to people.

[0116] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0117] I. Experimental Materials, Reagents, and Instruments

[0118] Escherichia coli DH5α, BL21 (DE3), gene site-directed mutagenesis kit, endonucleases Dpn I, Nde I, and Xho I, LB medium-related reagents, antibiotics, inducers, plasmid mini-extraction kit, Ni affinity chromatography column packing, ATP standard, firefly luciferase detection reagent, 96-well plate, and other materials and reagents are all commercially available from Shanghai Beyotime Biotechnology Co., Ltd.

[0119] The gene random mutagenesis kit was purchased from Takara.

[0120] The PCR instrument was purchased from Bio-Rad.

[0121] Ultrasonic cell disruptor was purchased from Ningbo Xinzhi Biotechnology Co., Ltd.

[0122] High-pressure cell disruptor was purchased from Antos Nanotechnology (Suzhou) Co., Ltd.

[0123] Clinx gel imager was purchased from Shanghai Qinxiang Scientific Instrument Co., Ltd.

[0124] The multifunctional microplate reader Varioskan LUX was purchased from ThermoFisher.

[0125] II. Experimental Methods

[0126] 1. Structure prediction and analysis of wild-type Pennsylvania firefly (Photuris pensylvanica) luciferase and Luc146-1H2 mutant

[0127] The three-dimensional structure of a protein can intuitively reveal the regions where the protein interacts with its substrate, the mechanisms of substrate binding and release, and key amino acid sites involved in substrate-specific recognition. This provides valuable guidance for targeted mutagenesis of firefly (Photuris pensylvanica) luciferase. Currently, there is no resolved spatial structural data for firefly (Photuris pensylvanica) luciferase. Using protein three-dimensional structure prediction tools, we predicted its overall structure and conducted key enzyme active site analysis, along with experimental analysis. Based on these results, we hypothesized amino acid sites that may be involved in substrate recognition and binding, and thus influence luciferase thermostability. Based on this, we constructed a series of site-directed mutants based on the Luc146-1H2 mutant. These mutants were then tested for enzyme activity and thermostability, hoping to identify mutants that significantly improve these two aspects. The protein sequences of the wild-type and Luc146-1H2 mutant firefly luciferases are shown in the "Sequence Information" section above.

[0128] 2. Construction of luciferase site-directed mutants

[0129] The 146-1H2 gene was obtained by gene synthesis (sequence see the "Sequence Information" section) and cloned into the pET22b expression vector through the NdeI and XhoI restriction sites. A 6X His purification tag and a stop codon were added to the C-terminus of the protein to obtain the pET22b-luc146-1H2 plasmid.

[0130] Then, using the pET22b-luc146-1H2 plasmid as a template, we designed and synthesized the gene site-directed mutagenesis PCR primers and used QuickMutation TM Gene site-directed mutagenesis PCR was performed using a gene site-directed mutagenesis kit (Biyuntian, D0206).

[0131] A 50 μl reaction system includes: 29 μl nuclease-free water, 5 μl 10X BeyoFusion TM Buffer (containing Mg 2+), 10 μl dNTP mix (2.5 mM each), 2 μl forward primer (10 μM), 2 μl reverse primer (10 μM), 1 μl pET22b-lucl46-1H2 plasmid (200 ng), and 1 μl BeyoFusion TM DNA polymerase.

[0132] The PCR program was as follows: pre-denaturation at 95°C for 3 min; 20 cycles of 95°C for 30 s, 55°C for 30 s, and 68°C for 3 min (30 s / kb); extension at 68°C for 15 min; and temporary storage at 4°C.

[0133] After the PCR reaction was completed, 1 μl of endonuclease Dpn I (Biyuntian, D6257) was added to the PCR reaction system, mixed well, and incubated at 37° C. for 1 hour to digest the template plasmid.

[0134] 10 μl of each digested PCR product was added to 100 μl of DH5α super competent cells (Biyuntian, D1031), mixed, placed on ice for 30 min, heat shocked at 42°C for 90 s, and incubated on ice for 2 min.

[0135] Add 900 μl of resistance-free LB medium and shake at 37°C for 1 hour. Collect the cells by centrifugation and resuspend them in 100 μl of resistance-free LB medium. Spread the culture evenly on an LB plate containing 100 μg / ml ampicillin (Biyuntian, ST007). Invert the plate in a 37°C incubator and culture overnight.

[0136] The next day, a single colony was picked and transferred to 10 ml of LB liquid medium containing 100 μg / ml ampicillin and cultured overnight. The plasmids in the bacterial suspension of each mutant were extracted using a plasmid miniprep kit (Biyuntian, D0003), and the plasmid concentration was determined.

[0137] Sequence the target gene using T7 and T7-Terminator universal primers. For mutants with multiple mutation sites, multiple rounds of site-directed mutagenesis are required, with one or more mutations introduced in each round. Each round requires sequencing verification to ensure success before proceeding to the next round.

[0138] 3. Construction of luciferase random mutants

[0139] In order to improve the performance of Luc146-1H2 mutants to the greatest extent possible and screen more high-quality mutants, we used the pET22b-luc146-1H2 plasmid as a template while performing site-directed mutagenesis. The PCR Random Mutagenesis Kit performs random gene mutation PCR, constructs random mutants, and screens the enzyme activity and thermal stability of random mutants.

[0140] The 50 μl reaction system for random mutation PCR included: 35 μl PCR Grade Water, 5 μl 10x TITANIUM Taq Buffer, 4 μl MnSO4 (8 mM), 1 μl dGTP (2 mM), 1 μl 50x Diversify dNTP Mix, 1 μl forward primer (10 μM, sequence: 5'-gtgctcgagttagtggtgatggtg-3' (SEQ ID NO: 6)), 1 μl reverse primer (10 μM, sequence: 5'-agatatacatatggcagataagaatattt-3' (SEQ ID NO: 7)), 1 μl pET22b-lucl46-1H2 plasmid (1 ng), and 1 μl TITANIUM Taq polymerase.

[0141] The PCR program was as follows: pre-denaturation at 94°C for 30 s; 25 cycles of 94°C for 30 s, 45°C for 30 s, and 68°C for 2 min; extension at 68°C for 1 min; and temporary storage at 4°C.

[0142] Enzyme digestion: 50 μl of the reaction system includes: PCR Grade Water, 5 μl 10x Buffer R, 1 μl NdeI (Biyuntian, D6485), 1 μl XhoI (Biyuntian, D6721), and 2 μg of PCR product. Incubate at 37°C overnight.

[0143] The overnight digestion product was ligated to the pET22b vector linearized by NdeI and XhoI digestion, and the ligation product was transformed into BL31 (DE3) competent cells.

[0144] Single clones were picked from the plate for protein expression and Bac-Lumi TM The bacterial firefly luciferase detection reagent (Biyuntian, RG039) was used to detect the enzyme activity of different mutant luciferase crude enzymes.

[0145] 4. Enzyme activity detection of crude luciferase mutants

[0146] Bac-Lumi TMThaw the bacterial firefly luciferase detection reagent (Beyotime, RG039) and equilibrate to room temperature. To avoid excessive signal values ​​that exceed the linear range of the microplate reader, dilute the induced bacterial solution 10 times with LB, and then take 100 μl to BeyoGold TM In a white 96-well cell culture plate (flat-bottomed, individually packaged) (Beyond Technology, FCP968), 100 μl of LB was used as a blank control. 100 μl of bacterial firefly luciferase detection reagent was added to each well. The 96-well plate was placed on a horizontal decolorization shaker and incubated at room temperature for 10 minutes. Chemiluminescence was then measured using a multi-functional microplate reader, with readings taken every 2 minutes for a total of 2 hours. The RLU (reduced unit) chemiluminescence signal roughly reflects the enzyme activity of the different mutants.

[0147] 5. Expression of Firefly Luciferase Mutants with High Enzyme Activity

[0148] Escherichia coli BL21(DE3) glycerol strains (Beyotime, D0337) were prepared into competent cells using a super competent bacteria preparation kit (Beyotime, D0302). Correctly sequenced mutants, wild-type, and control plasmids were transformed into E. coli BL21(DE3) competent cells and cultured overnight at 37°C on LB plates containing ampicillin. The next day, a single colony was picked and transferred to 10 ml of LB liquid medium containing ampicillin. The cells were cultured in a shaker at 37°C, 220 rpm, until the OD600 value was between 0.6 and 0.8. IPTG (Beyotime, ST098) was added to a final concentration of 0.1 mM and cultured at 18°C, 220 rpm, for 16 hours to induce protein expression. 10 ml of the bacterial suspension before and after induction was collected by centrifugation and resuspended in 10 ml of PBS. The cells were disrupted in an ultrasonic cell disruptor for 10 minutes and then centrifuged at 12,000 rpm for 10 minutes. Take 50 μl of the supernatant after centrifugation and add 10 μl of 6X SDS-PAGE protein loading buffer (Biyuntian, P0015F). Heat in a boiling water bath at 100°C for 10 minutes and centrifuge at 12,000 rpm for 10 minutes. Take 20 μl of each supernatant and perform SDS-PAGE electrophoresis to observe the expression of firefly luciferase protein.

[0149] 6. Purification of Firefly Luciferase Mutants with High Enzyme Activity

[0150] According to the results of crude enzyme activity detection, 23 firefly luciferase mutants with significantly improved enzyme activity were screened and expressed in 1L of each in BL21 (DE3) strain together with the control (146-1H2). The following purification steps were all carried out at 4°C. The induced bacteria were collected and resuspended in 100ml lysis buffer (50mM NaH2PO4 pH8.0, 300mM NaCl, 10% glycerol, 1mM PMSF) and crushed 3 times in a high-pressure cell crusher at a pressure of 850bar. After centrifugation at 12000rpm for 30min, the supernatant was collected and precipitated using BeyoGold TM The target protein was purified using a His-tag Purification Resin (reduction-resistant chelating type) (Biyuntian, P2218) affinity purification column. The purified firefly luciferase was dialyzed into enzyme storage solution (25mM Tris-acetate pH 7.8, 1mM EDTA, 1mM DTT, 0.2M (NH4)2SO4, 15% glycerol, 30% ethylene glycol). Protein concentration was determined using a Bradford Protein Assay Kit (detergent-compatible) (Biyuntian, P0006) and confirmed by SDS-PAGE. Aliquoted protein was snap-frozen in liquid nitrogen and stored at -80°C.

[0151] 7. Quantitative detection of enzyme activity and ATP of purified firefly luciferase mutants

[0152] Remove the existing product CellTiter-Lumi TM A new detection reagent, abbreviated as CTL, was obtained by using the luciferase component of the luminescent cell viability assay kit (Biyuntian, C0065). Firefly luciferase mutants and controls were diluted to 1 mg / ml using CTL, and ATP standards (Biyuntian, D7378) were diluted to 2, 1.5, 1, 0.5, 0.25, and 0.125 μM using PBS. A 200 μl reaction system was prepared in a 96-well plate: 5 μl of 1 mg / ml firefly luciferase protein, 95 μl of CTL, and 100 μl of ATP standards of varying concentrations (0-2 μM) (added last). The 96-well plate was placed on a horizontal decolorization shaker and incubated at room temperature for 10 minutes. Chemiluminescence was then measured using a multi-function microplate reader, with readings taken every minute for a total of 20 minutes.

[0153] 8. Thermal stability test of purified firefly luciferase mutants

[0154] Dilute the firefly luciferase mutant and control to 1 mg / ml using enzyme stock solution. Place 100 μl of each aliquot (50 μl x 2) in a PCR instrument and incubate at 50°C for 24 hours. After incubation, centrifuge at 12,000 rpm for 10 minutes, remove the supernatant, and measure enzyme activity simultaneously with the unheated protein. Compare the enzyme activity of heated and unheated firefly luciferase.

[0155] 9. Purified firefly luciferase mutants are used to detect cell viability

[0156] Purified M1 and M2 were used to measure the intracellular ATP content of different numbers of HeLa cells to reflect cell viability. Cultured HeLa cells were diluted proportionally with culture medium to form a series of concentration gradients and incubated with 1% Triton X-100 to completely lyse the HeLa cells. In a 96-well plate for chemiluminescence detection, 100 μl of HeLa cells at different concentrations were seeded into each well, so that each reaction system contained 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10,000, 20,000, and 50,000 cells, respectively. Wells containing culture medium without cells were also set up as negative controls for measuring intracellular ATP content. Cell viability was assessed by performing a luminescence reaction in 200 μl of a luciferase mutant at a final concentration of 25 μg / ml.

[0157] 10. Sequence information

[0158] M3 (Q73H) amino acid sequence:

[0159] Based on SEQ ID NO: 2, position 73 was mutated from Q to H.

[0160] M4 (G158E, N402S, Y462N) amino acid sequence:

[0161] Based on SEQ ID NO: 2, position 158 was mutated from G to E, position 402 was mutated from N to S, and position 462 was mutated from Y to N.

[0162] M5 (V239A) amino acid sequence:

[0163] Based on SEQ ID NO: 2, position 239 was mutated from V to A.

[0164] M6(K379R) amino acid sequence:

[0165] Based on SEQ ID NO: 2, position 379 was mutated from K to R.

[0166] M7 (I145V, M248K, T351S, K532I) amino acid sequence:

[0167] Based on SEQ ID NO: 2, position 145 was mutated from I to V, position 248 was mutated from M to K, position 351 was mutated from T to S, and position 532 was mutated from K to I.

[0168] Among them, the wild-type position 145 is V, and I145V is equivalent to the residue mutated back to the corresponding position of WT.

[0169] M8 (N130S, K212E) amino acid sequence:

[0170] Based on SEQ ID NO: 2, position 130 was mutated from N to S, and position 212 was mutated from K to E.

[0171] M9 (F293L, V365I, G378V) amino acid sequence:

[0172] Based on SEQ ID NO: 2, position 293 was mutated from F to L, position 365 was mutated from V to I, and position 378 was mutated from G to V.

[0173] M10 (M208T, I311F, E321G, L538F) amino acid sequence:

[0174] Based on SEQ ID NO: 2, position 208 was mutated from M to T, position 311 was mutated from I to F, position 321 was mutated from E to G, and position 538 was mutated from L to F.

[0175] M11 (N74K) amino acid sequence:

[0176] Based on SEQ ID NO: 2, position 74 was mutated from N to K.

[0177] M12 (Q277L, K379R, N413S, T488S, N543Y) amino acid sequence:

[0178] Based on SEQ ID NO: 2, position 277 was mutated from Q to L, position 379 was mutated from K to R, position 413 was mutated from N to S, position 488 was mutated from T to S, and position 543 was mutated from N to Y.

[0179] Among them, position 413 of WT is K, and position 413 of 146-1H2 is K413N, where N413 mutated to S.

[0180] Position 543 of WT is S, and position 543 of 146-1H2 is S543N, where N543 is mutated to Y.

[0181] M13 (N137K, A235T, K280R, N333D, 1422V) amino acid sequence:

[0182] Based on SEQ ID NO: 2, position 137 was mutated from N to K, position 235 was mutated from A to T, position 280 was mutated from K to R, position 333 was mutated from N to D, and position 422 was mutated from I to V.

[0183] M14(K57R) amino acid sequence:

[0184] Based on SEQ ID NO: 2, position 57 was mutated from K to R.

[0185] M15 (T42A, E282D, K392I, D426V) amino acid sequence:

[0186] Based on SEQ ID NO: 2, position 42 was mutated from T to A, position 282 was mutated from E to D, position 392 was mutated from K to I, and position 426 was mutated from D to V.

[0187] M16 (E110G, V281E, I364V, E404G) amino acid sequence:

[0188] Based on SEQ ID NO: 2, position 110 was mutated from E to G, position 281 was mutated from V to E, position 364 was mutated from I to V, and position 404 was mutated from E to G.

[0189] M17 (S525P) amino acid sequence:

[0190] Based on SEQ ID NO: 2, position 525 was mutated from S to P.

[0191] M18 (E64V, N186S) amino acid sequence:

[0192] Based on SEQ ID NO: 2, position 64 was mutated from E to V, and position 186 was mutated from N to S.

[0193] M19 (V79M, D374N, Q536R) amino acid sequence:

[0194] Based on SEQ ID NO: 2, position 79 was mutated from V to M, position 374 was mutated from D to N, and position 536 was mutated from Q to R.

[0195] M20 (N231K) amino acid sequence:

[0196] Based on SEQ ID NO: 2, position 231 was mutated from N to K.

[0197] M21 (N231R) amino acid sequence:

[0198] Based on SEQ ID NO: 2, position 231 was mutated from N to R.

[0199] M22 (N231A) amino acid sequence:

[0200] Based on SEQ ID NO: 2, position 231 was mutated from N to A.

[0201] M23 (A229R) amino acid sequence:

[0202] Based on SEQ ID NO: 2, position 229 was mutated from A to R.

[0203] M24 (A229K) amino acid sequence:

[0204] Based on SEQ ID NO: 2, position 229 was mutated from A to K.

[0205] M25 (A229K, N231K) amino acid sequence:

[0206] Based on SEQ ID NO: 2, position 229 was mutated from A to K, and position 231 was mutated from N to K.

[0207] M26 (A229R, N231K) amino acid sequence:

[0208] Based on SEQ ID NO: 2, position 229 was mutated from A to R, and position 231 was mutated from N to K.

[0209] M27 (N231K, F293L) amino acid sequence:

[0210] Based on SEQ ID NO: 2, position 231 was mutated from N to K, and position 293 was mutated from F to L.

[0211] M28 (A229K, N231R) amino acid sequence:

[0212] Based on SEQ ID NO: 2, position 229 was mutated from A to K, and position 231 was mutated from N to R.

[0213] M29 (A229R, N231R) amino acid sequence:

[0214] Based on SEQ ID NO: 2, position 229 was mutated from A to R, and position 231 was mutated from N to R.

[0215] M30 (N231R, F293L) amino acid sequence:

[0216] Based on SEQ ID NO: 2, position 231 was mutated from N to R, and position 293 was mutated from F to L.

[0217] M31 (A229K, N231A) amino acid sequence:

[0218] Based on SEQ ID NO: 2, position 229 was mutated from A to K, and position 231 was mutated from N to A.

[0219] M32 (A229R, N231A) amino acid sequence:

[0220] Based on SEQ ID NO: 2, position 229 was mutated from A to R, and position 231 was mutated from N to A.

[0221] M33 (N231A, F293L) amino acid sequence:

[0222] Based on SEQ ID NO: 2, position 231 was mutated from N to A, and position 293 was mutated from F to L.

[0223] M34 (A229K, N231K, F293L) amino acid sequence:

[0224] Based on SEQ ID NO: 2, position 229 was mutated from A to K, position 231 was mutated from N to K, and position 293 was mutated from F to L.

[0225] M35 (A229R, N231K, F293L) amino acid sequence:

[0226] Based on SEQ ID NO: 2, position 229 was mutated from A to R, position 231 was mutated from N to K, and position 293 was mutated from F to L.

[0227] M36 (A229K, N231R, F293L) amino acid sequence:

[0228] Based on SEQ ID NO: 2, position 229 was mutated from A to K, position 231 was mutated from N to R, and position 293 was mutated from F to L.

[0229] M37 (A229R, N231R, F293L) amino acid sequence:

[0230] Based on SEQ ID NO: 2, position 229 was mutated from A to R, position 231 was mutated from N to R, and position 293 was mutated from F to L.

[0231] M38 (A229K, N231A, F293L) amino acid sequence:

[0232] Based on SEQ ID NO: 2, position 229 was mutated from A to K, position 231 was mutated from N to K, and position 293 was mutated from F to L.

[0233] Example 1. Prediction of the three-dimensional structure of firefly (Photuris pensylvanica) luciferase and comparison of its overall structure.

[0234] Using protein structure prediction tools, the overall spatial structure of firefly (Photuris pensylvanica) luciferase was successfully predicted. To further explore and analyze the binding mechanism between luciferase and its substrate, the predicted structure was compared with the previously published structure of a luciferase-substrate complex in the PDB database (PDB ID: 5dwv).

[0235] The results are shown in Figure 1: Alignment of the firefly (Photuris pensylvanica) luciferase structure with the 5dwv structure from two different orientations. The results show that the two share the same overall topological conformation, with a high degree of structural similarity (RMSD = 0.0731), and the spatial regions responsible for substrate binding are consistent.

[0236] Example 2: Analysis of the three-dimensional structure of firefly (Photuris pensylvanica) luciferase and display of the substrate binding pocket.

[0237] Based on the structure, one luciferase molecule binds to one substrate molecule. The substrate binds within a hydrophobic pocket formed by one α-helix, one loop, and four β-sheets. Several amino acids located near this hydrophobic pocket, through their side chains, provide effective interactions for substrate anchoring, stabilizing specific substrate recognition and binding. Furthermore, the side chains of amino acids located at the entrance and exit of the substrate-binding pocket seal the pocket, collectively preventing the escape and release of the bound substrate.

[0238] The results are shown in Figure 2: Detailed representation of the substrate binding pocket. The overall three-dimensional structure of firefly (Photuris pensylvanica) luciferase is shown on the left. Local details of the luciferase-substrate interaction are shown on the right.

[0239] Example 3. Construction of mutants and expression of luciferase

[0240] Based on the results of structural analysis, a series of site-directed mutagenesis sites were designed for luciferase. The site-directed mutagenesis PCR product was digested with Dpn I and transformed into competent E. coli DH5α cells. Three single clones were selected for small-scale culture and plasmid extraction. Sequence verification was performed to identify clones that successfully achieved site-directed mutagenesis.

[0241] Ultimately, all designed site-directed mutants obtained positive clones, and the plasmid sequences were verified by sequencing to ensure their correctness; while random mutants were cloned by direct screening and sequencing verification using a random mutagenesis kit.

[0242] 38 firefly luciferase mutants and the control WT and 146-1H2 were significantly expressed in the supernatant.

[0243] Example 4. Enzyme activity assay of wild-type, 146-1H2 and mutant crude enzymes

[0244] Bac-Lumi TM The Bacterial Firefly Luciferase Detection Reagent contains both E. coli cell lysis components and firefly luciferase activity detection components, enabling a one-step determination of bacterial firefly luciferase activity without the need for cell harvesting and lysis. In a 96-well plate, mix 100 μl of a 10-fold diluted induced bacterial solution with 100 μl of the detection reagent at a 1:1 ratio. Incubate on a horizontal shaker at room temperature for 10 minutes, then measure chemiluminescence using a multi-functional microplate reader. Graph the fluorescence signal after 1 hour of reaction (the WT signal decays rapidly, so the signal value at 10 minutes is used for graphing).

[0245] The results are shown in Figure 3: The signal value of the wild type (WT) is 1.7×10 3 RLU, the signal of the control (146-1H2) was approximately 1.7×10 8 RLU, the enzyme activity signal of M1-M38 ranged from 8.1×10 7 -4.8×10 8 Between RLUs.

[0246] Example 5. Expression, purification and quantification of firefly luciferase proteins with significant effects in crude enzyme activity detection

[0247] Based on the crude enzyme activity assay results, 23 mutants with relatively high enzyme activity were obtained. 1 L of each mutant and control (WT, 146-1H2) were expressed in the BL21(DE3) strain and affinity purified using a Ni affinity column. The purified proteins were then diluted to the same concentration of 1 mg / ml using enzyme stock solution. SDS-PAGE was then performed to confirm the consistency of the 25 luciferase protein samples.

[0248] The results are shown in Figure 4: the loading volume of each sample was 1 μg, and the protein amount among the samples was basically consistent.

[0249] Example 6. Comparison of Enzyme Activity Detection of Purified Firefly Luciferase

[0250] In this example, each luciferase mutant diluted to 1 mg / ml with enzyme stock solution was used for enzyme activity detection.

[0251] In a 200 μl reaction system, the final protein concentration of various firefly luciferase mutants and controls (WT, 146-1H2) was 25 μg / ml, and the signal value at the 10th minute of detection was plotted.

[0252] The results are shown in Figure 5. Compared to the control (146-1H2), the enzyme activities of the remaining 23 firefly luciferase mutants were significantly increased, approximately 1.6-2.8 times that of the control. This enzyme activity test result is basically consistent with the previous crude enzyme activity test results.

[0253] Example 7. Comparison of signal thermal stability of purified firefly luciferase

[0254] In this example, each luciferase mutant diluted to 1 mg / ml with enzyme stock solution was used for enzyme activity detection.

[0255] In a 200 μl reaction system, the final protein concentration of various firefly luciferase mutants and controls (WT, 146-1H2) was 25 μg / ml. Firefly luciferase protein samples with standardized protein concentrations were treated before and after incubation at 50°C, and then enzyme activity was measured. The thermal stability of each luciferase mutant was analyzed based on the change in enzyme activity before and after incubation at 50°C.

[0256] The results are shown in Figure 6: After incubation at 50°C, the enzymatic activity of the wild-type luciferase was almost completely lost; the enzymatic activity of the luciferase mutant 146-1H2 decreased slightly, about 10% lower than before incubation; the enzymatic activity of the remaining luciferase mutants decreased to varying degrees, ranging from 1-27%, after incubation at 50°C, and all mutants showed high or very high thermal stability.

[0257] Example 8: Firefly luciferase mutants with high enzyme activity are used for quantitative detection of ATP

[0258] Based on the results of the previous tests, two firefly luciferase mutants, M1 (F293L) and M2 (Y254S), were selected to test whether they could be used for the quantitative detection of ATP.

[0259] In a 200 μl reaction system, the final protein concentration of firefly luciferase mutants M1 (F293L), M2 (Y254S) and the control (146-1H2) was 25 μg / ml; the final ATP concentration was 0, 0.125, 0.25, 0.5, 1, 1.5, and 2 μM.

[0260] The signal values ​​at 10 minutes after detection were plotted, and the results are shown in FIG7 : the fluorescence signal values ​​of the M1 (F293L) and M2 (Y254S) mutants were significantly higher than those of the control (146-1H2).

[0261] The signal values ​​of the mutants at various ATP concentrations (excluding 0 μM) were calculated as a multiple of the control. The enzyme activity of M1 (F293L) was approximately 2.82 ± 0.09 times that of the control (146-1H2), and the enzyme activity of M2 (Y254S) was approximately 2.72 ± 0.05 times that of the control (146-1H2); that is, the enzyme activities of the mutants M1 (F293L) and M2 (Y254S) were approximately 182% and 172% higher than those of the control (146-1H2), respectively.

[0262] Example 9: Firefly luciferase mutants with high enzyme activity can still be used for quantitative detection of ATP after heating at 50 degrees

[0263] 1 mg / ml firefly luciferase samples were heat-treated at 50°C for 24 hours, then centrifuged and the supernatant was collected. The enzyme activity of the supernatant was compared with that of the unheat-treated firefly luciferase. The signal value at the 10th minute of detection was plotted.

[0264] The results are shown in Figure 8. All sample signal values ​​showed a positive correlation with ATP concentration, indicating that mutants M1(F293L) and M2(Y254S), like the control (146-1H2), can be used to detect ATP concentration in solution. Overall, the enzyme activity of M1(F293L) was approximately 2.8 times that of the control (146-1H2), while that of M2(Y254S) was nearly 2.7 times that of the control (146-1H2).

[0265] This result is consistent with that in Figure 6, indicating that the experiment has good repeatability.

[0266] Comparing the data of the same protein before and after heat treatment, it was found that treatment at 50°C for 24 hours did not affect the enzyme activity of the protein, indicating that M1 (F293L) and M2 (Y254S) have good thermal stability.

[0267] Example 10: Comparison of the effects of high-activity firefly luciferase mutants in cellular enzyme activity detection

[0268] Purified firefly luciferase mutants M1 and M2 with high enzymatic activity were used to measure ATP content in HeLa cells of varying numbers using a chemiluminescent assay to assess cell viability, achieving ultra-high sensitivity and a wide linear range for quantitative determination of viable cell numbers. ATP concentrations were measured after cells were lysed with 1% Triton X-100. Signal values ​​at the 10th minute of detection were plotted.

[0269] The results are shown in Figure 9: Luciferase mutants can be used for cell enzyme activity detection with high sensitivity and a wide linear range, showing a good linear relationship at least in the range of 0 to 50,000 cells.

[0270] Experimental Conclusion

[0271] Through structural prediction and comparative analysis, combined with site-directed and random mutagenesis, and in-depth analysis and experimental research, the inventors designed and screened a series of firefly luciferase mutants. After initial screening, 23 mutants with high enzyme activity were expressed, purified, and assayed for enzyme activity.

[0272] Testing revealed that all 23 purified mutants exhibited significantly increased enzyme activity compared to the control (146-1H2). After 24 hours of incubation at 50°C, all 23 mutants retained significant enzyme activity. M1 (F293L) and M2 (Y254S) exhibited relatively good enzyme activity and thermal stability. These two mutants, selected as examples, were found to be highly suitable for quantitative ATP detection, and both mutants remained well-suited for ATP quantification even after 24 hours of incubation at 50°C. Furthermore, these mutants were found to be useful for measuring cell number or viability.

[0273] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

Claims

1. A method for improving the enzymatic activity and / or thermal stability of firefly luciferase, comprising: Firefly luciferase is mutated and transformed to form a firefly luciferase mutant, which is adjacent to the substrate binding pocket in spatial structure and contributes to the recognition and binding of the substrate or the stabilization of the spatial conformation of the protein.

2. [Corrected 12.08.2024 in accordance with Rule 26] A method as claimed in claim 1, characterized in that Corresponding to the amino acid sequence shown in SEQ ID NO: 2, the following mutations were performed: A. A mutation selected from (a)-(t) or a combination thereof: (a) The amino acid F at position 293 mutated to L (M1); (b) amino acid Y at position 254 mutated to S (M2); (c) position 239 mutated from V to A (M5); (d) position 137 mutated from N to K, position 235 mutated from A to T, position 280 mutated from K to R, position 333 mutated from N to D, and position 422 mutated from I to V (M13); (e) position 57 mutated from K to R (M14); (f) position 42 mutated from T to A, position 282 mutated from E to D, position 392 mutated from K to I, and position 426 mutated from D to V (M15); (g) position 525 mutated from S to P (M17); (h) amino acid N at position 231 is mutated to A, R or K (M20, M21 or M22); (i) amino acid A at position 229 is mutated to R or K (M23 or M24); (j) position 229 mutated from A to K, and position 231 mutated from N to K (M25); (k) position 231 mutated from N to K, and position 293 mutated from F to L (M27); (l) position 229 mutated from A to R, and position 231 mutated from N to R (M29); (m) position 231 mutated from N to R, and position 293 mutated from F to L (M30); (n) position 229 mutated from A to K, and position 231 mutated from N to A (M31); (o) position 231 mutated from N to A, and position 293 mutated from F to L (M33); (p) position 229 mutated from A to K, position 231 mutated from N to K, and position 293 mutated from F to L (M34); (q) position 229 mutated from A to R, position 231 mutated from N to K, and position 293 mutated from F to L (M35); (r) position 229 mutated from A to K, position 231 mutated from N to R, and position 293 mutated from F to L (M36); (s) position 229 mutated from A to R, position 231 mutated from N to R, and position 293 mutated from F to L (M37); (t) position 229 mutated from A to K, position 231 mutated from N to K, and position 293 mutated from F to L (M38); B. A protein derived from A which is formed by replacing, deleting or adding one or more amino acid residues in the amino acid sequence of protein A and has the function of protein A, but the sites corresponding to (a)-(t) in A are conserved; C. A protein derived from A that has more than 80% homology with the amino acid sequence of A protein and has the function of A protein, but the sites defined by (a)-(t) in A are conserved.

3. A firefly luciferase mutant, characterized in that: Its spatial structure is adjacent to the substrate binding pocket, and contributes to the recognition and binding of the substrate or the stability of the spatial conformation of the protein. Its amino acid sequence corresponds to the amino acid sequence shown in SEQ ID NO: 2, and the following mutations are performed: A. A mutation selected from (a)-(t) or a combination thereof: (a) The amino acid F at position 293 mutated to L (M1); (b) amino acid Y at position 254 mutated to S (M2); (c) position 239 mutated from V to A (M5); (d) position 137 mutated from N to K, position 235 mutated from A to T, position 280 mutated from K to R, position 333 mutated from N to D, and position 422 mutated from I to V (M13); (e) position 57 mutated from K to R (M14); (f) position 42 mutated from T to A, position 282 mutated from E to D, position 392 mutated from K to I, and position 426 mutated from D to V (M15); (g) position 525 mutated from S to P (M17); (h) amino acid N at position 231 is mutated to A, R or K (M20, M21 or M22); (i) amino acid A at position 229 is mutated to R or K (M23 or M24); (j) position 229 mutated from A to K, and position 231 mutated from N to K (M25); (k) position 231 mutated from N to K, and position 293 mutated from F to L (M27); (l) position 229 mutated from A to R, and position 231 mutated from N to R (M29); (m) position 231 mutated from N to R, and position 293 mutated from F to L (M30); (n) position 229 mutated from A to K, and position 231 mutated from N to A (M31); (o) position 231 mutated from N to A, and position 293 mutated from F to L (M33); (p) position 229 mutated from A to K, position 231 mutated from N to K, and position 293 mutated from F to L (M34); (q) position 229 mutated from A to R, position 231 mutated from N to K, and position 293 mutated from F to L (M35); (r) position 229 mutated from A to K, position 231 mutated from N to R, and position 293 mutated from F to L (M36); (s) position 229 mutated from A to R, position 231 mutated from N to R, and position 293 mutated from F to L (M37); (t) position 229 mutated from A to K, position 231 mutated from N to K, and position 293 mutated from F to L (M38); B. A protein derived from A which is formed by replacing, deleting or adding one or more amino acid residues in the amino acid sequence of protein A and has the function of protein A, but the sites corresponding to (a)-(t) in A are conserved; C. A protein derived from A that has more than 80% homology with the amino acid sequence of A protein and has the function of A protein, but the sites defined by (a)-(t) in A are conserved.

4. A polynucleotide, characterized in that The polynucleotide encodes the firefly luciferase mutant according to claim 3.

5. A vector or a genetically engineered host cell containing the vector, characterized in that: The vector contains the polynucleotide according to claim 4; or the host cell contains the vector or the polynucleotide according to claim 4 is integrated into its genome.

6. A method for producing the mutant of firefly luciferase according to claim 3, characterized in that: Includes steps: (i) culturing the host cell according to claim 5; (ii) collecting a culture containing the firefly luciferase mutant; (iii) isolating the firefly luciferase mutant from the culture.

7. Use of the firefly luciferase mutant according to claim 3, the host cell expressing the mutant or its lysate, for catalyzing the oxidation of firefly luciferase substrates, generating ATP-dependent bioluminescence, and detecting cell number or cell viability.

8. A method for catalytically oxidizing firefly luciferin to produce bioluminescence, comprising: The firefly luciferase mutant according to claim 3, a host cell expressing the mutant or a lysate thereof is used for catalytic oxidation to produce bioluminescence in the presence of ATP.

9. The method according to claim 8, characterized in that Used for reactions including the following groups: in vitro ATP assay, cell viability assay, cell number assay.

10. The method according to claim 9, characterized in that The in vitro ATP detection comprises: using the firefly luciferase mutant and the luciferase substrate to measure the amount of ATP contained in the solution system; or The cell viability detection comprises: using the firefly luciferase mutant and luciferase substrate, mixing with cell culture, and measuring cell viability; or The cell number detection comprises: utilizing the firefly luciferase mutant and luciferase substrate, mixing them with cell culture, and determining the cell number.

11. A detection system or detection kit for catalyzing the oxidation of firefly luciferin and producing bioluminescence, comprising: the firefly luciferase mutant according to claim 3, or the host cell or its culture or lysate according to claim 5.

12. An ATP tester, comprising: A container, comprising the firefly luciferase mutant according to claim 3; a container, a luciferase substrate contained in the container; The sampling, adding and reaction device is used to add the sample to be tested, and the sample to be tested is the sample that needs to be tested for ATP determination.

13. The ATP tester according to claim 12, characterized in that: A gas supply is also included, the gas comprising oxygen.

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