CN116814568A - Firefly luciferase mutant, protein, nucleic acid, recombinant vector, recombinant bacterium, reagent composition and preparation method - Google Patents
Firefly luciferase mutant, protein, nucleic acid, recombinant vector, recombinant bacterium, reagent composition and preparation method Download PDFInfo
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- CN116814568A CN116814568A CN202311058580.5A CN202311058580A CN116814568A CN 116814568 A CN116814568 A CN 116814568A CN 202311058580 A CN202311058580 A CN 202311058580A CN 116814568 A CN116814568 A CN 116814568A
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Abstract
The application relates to the technical field of biology, and particularly discloses firefly luciferase mutants, proteins, nucleic acids, recombinant vectors, recombinant bacteria, reagent compositions and preparation methods thereof. According to the application, the firefly luciferase gene is subjected to random mutation, and the luminous intensity and half-life period are screened, so that the firefly luciferase mutant with high activity and high stability is obtained through successful screening, and the method and the kit for detecting living cells by using an ATP detection means are further optimized.
Description
Technical Field
The application relates to the field of biotechnology, in particular to a firefly luciferase mutant, protein, nucleic acid, recombinant vector, recombinant bacterium, reagent composition and a preparation method thereof.
Background
Firefly luciferases are proteases capable of catalyzing and causing bioluminescence of firefly luciferin substrates in the presence of Adenosine Triphosphate (ATP), divalent metal ions such as magnesium ions and oxygen. Because of its unique bioluminescence characteristics and non-radioactive advantages, it is widely used in prokaryotic and eukaryotic cells, transgenic plants and animals, and cell-free detection systems.
ATP is the most direct source of energy in organisms, and detecting and quantifying intracellular ATP levels is the method of choice for assessing cellular health. The luciferase-luciferin detection system is widely applied to the field of drug development and detection of microbial contamination ATP in the environment. With the rapid development of biotechnology and the maturation of high throughput drug screening technologies, more efficient cell viability assay systems are needed.
Disclosure of Invention
In order to improve activity, stability and accuracy, the application provides firefly luciferase mutants, proteins, nucleic acids, recombinant vectors, recombinant bacteria, reagent compositions and preparation methods.
In a first aspect, the present application provides a firefly luciferase mutant, which adopts the following technical scheme:
a firefly luciferase mutant, wherein the firefly luciferase has an amino acid sequence shown in SEQ ID NO. 1, and is mutated at a position selected from the group consisting of: mutation of serine Ser S at 193 to isoleucine Ile I or mutation of leucine Leu L at 367 to isoleucine Ile I, as shown in SEQ ID No. 2 or SEQ ID No. 3 in the sequence list.
In a second aspect, the present application provides a protein, which adopts the following technical scheme:
a protein which is any one of the following (a 1) to (a 3):
(a1) An enzyme mutated at the 193 rd and 367 th positions of the amino acid sequence shown in SEQ ID NO. 1;
(a2) An enzyme derived from (a 1) having the function/activity of the enzyme of (a 1) and formed by substitution and/or deletion and/or addition of an amino acid residue at amino acid residues 193 and/or 367 in the amino acid sequence of the enzyme of (a 1);
the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues, or a substitution and/or deletion and/or addition of not more than 9 amino acid residues, or a substitution and/or deletion and/or addition of not more than 8 amino acid residues, or a substitution and/or deletion and/or addition of not more than 7 amino acid residues, or a substitution and/or deletion and/or addition of not more than 6 amino acid residues, or a substitution and/or deletion and/or addition of not more than 5 amino acid residues, or a substitution and/or deletion and/or addition of not more than 4 amino acid residues, or a substitution and/or deletion and/or addition of not more than 3 amino acid residues, or a substitution and/or deletion and/or addition of not more than 2 amino acid residues, or a substitution and/or deletion and/or addition of not more than 1 amino acid residue.
(a3) A polypeptide formed by connecting the N end or/and the C end of the mutated amino acid sequence of the (a 1) or the (a 2) with a tag sequence or an enzyme cutting site sequence, and a fusion protein. The tag refers to a polypeptide or protein which is fused and expressed together with a target protein by using a DNA in-vitro recombination technology so as to facilitate the expression, detection, tracing and/or purification of the target protein. The tag may be a Flag tag, a His tag, an HA tag, a GST tag, and/or a SUMO tag, etc.
The protein of (a 2) or (a 3) may be synthesized artificially or may be obtained by artificially synthesizing a nucleic acid molecule encoding the protein and expressing the resulting product in a host.
In another aspect of the application, any of the proteins described above has an enzymatic function/activity, including at least 75% (e.g., 75%,80%,85%,90%,95%, 100%) of its luminescent activity as compared to the activity of the enzyme of SEQ ID NO. 1.
In a third aspect, the present application provides a nucleic acid molecule, which adopts the following technical scheme:
a nucleic acid molecule comprising the nucleic acid molecule of (b 1) or (b 2) below:
(b1) The nucleotide sequence is a DNA molecule shown as encoding the protein;
(b2) A DNA molecule having at least 95% (e.g., 95%, 100%) identity to the nucleotide sequence of (b 1) and encoding the protein;
wherein the nucleic acid molecule can be DNA or RNA, and the nucleic acid molecule is formed by a gene encoding the protein and a regulatory sequence thereof.
In a fourth aspect, the present application provides a recombinant vector, which adopts the following technical scheme:
a recombinant vector comprising the above nucleic acid molecule. Further, the recombinant vector is a prokaryotic expression vector pET-28a (+).
In a fifth aspect, the present application provides a recombinant bacterium, which adopts the following technical scheme:
a recombinant bacterium comprising the recombinant vector or genome described above, wherein the nucleic acid molecule is integrated into the genome, and wherein the expression bacterium comprises a bacterium or fungus. Further, the bacterium may be E.coli BL21 (DE 3), including a recombinant bacterium obtained by introducing a recombinant vector encoding the above protein into E.coli.
In another aspect of the application, a method of preparing the above protein is also provided. Further, the method for preparing the protein provided by the application preferably comprises the following steps: the recombinant vector is transformed into host bacteria, and the recombinant bacteria are fermented and cultured in LB liquid medium containing kana antibiotics until the OD600 nm=0.8, 0.5mM IPTG is added, and the low temperature induction is carried out at 16 ℃ and 180rpm/min for overnight.
In another aspect of the present application, there is provided a reagent composition and/or kit comprising the above protein, corresponding to firefly luciferase as shown in SEQ ID NO:1, mutated at a site or combination of sites selected from the group consisting of: 193 rd bit or 367 th bit.
In a sixth aspect, the present application provides a method for preparing a firefly luciferase mutant, which adopts the following technical scheme:
a preparation method of firefly luciferase mutant comprises the steps of taking an artificially synthesized SEQ ID NO:1 nucleotide sequence as a template, establishing a random mutant library through a random mutation kit, cloning the mutant library into a prokaryotic expression vector, further transferring BL21 (DE 3) competent cells to induce expression of the mutant library, cracking a culture, detecting, calculating the RLU value of each mutant, and selecting a sample with the highest ratio to obtain the firefly luciferase.
In a seventh aspect, the present application provides a reagent composition, which adopts the following technical scheme:
a reagent composition comprising the firefly luciferase mutant protein described above and a firefly luciferin substrate.
Also comprises magnesium ions including magnesium sulfate, magnesium chloride, magnesium gluconate, magnesium acetate, magnesium bromide, magnesium carbonate and the like. Further, in any case, the magnesium complex must be dissociated to make magnesium ions available to the luciferase without interfering with the luciferase-luciferin reaction.
Also contains one or more additional components, including: buffers, salts, protease inhibitors, cell lysing agents, detergents, atpase inhibitors, ATP producing enzyme inhibitors, ATP extracting agents, conA, thiol reagents, metal ion chelating agents, and the like.
The additional ingredients include one or more suitable buffers. Any buffer that does not significantly interfere with the luciferase-luciferin reaction, i.e., a pH of a suitable working solution, may be considered, with a preferred pH range being between about pH 4.5 and about pH 9.0 (e.g., about pH 6.0 and about pH 8.0 (e.g., 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 and ranges therebetween)). Suitable buffers include MES, citrate buffer, phosphate Buffer (PBS), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), piperazine-1, 4-diethylsulfonic acid (PIPES), borates, and any other buffer as may be suitable as will be appreciated by one of skill in the art.
The additional ingredients include one or more defoamers therein. Defoamers to prevent system foam from interfering with detection of bioluminescence, particularly in applications where luminescence is quantified. Defoamers, such as MAZU, may be organic or silicone based. The choice of defoamers depends on their ability to eliminate foam without interfering with the luciferase-luciferin reaction.
Additional ingredients include one or more atpase inhibitor ingredients, optionally including or replacing other functional ingredients in the solution, such as buffers, defoamers, and the like. The assembly may be provided as a working solution or concentrate. Further, ATPase inhibitors are charged group detergents (e.g., cationic detergents (e.g., DTAB (dodecyltrimethylammonium bromide), CTAB (cetyltrimethylammonium bromide), benzalkonium chloride, benzalkonium bromide, etc.),
anionic detergents (e.g., deoxycholate or SDS) or zwitterionic detergents (e.g., dodecyl dimethyl betaine, etc.)).
The additional components include an ATP producing enzyme inhibitor. Further, ATP producing enzyme inhibitors include sodium fluoride, which is useful at concentrations ranging between at least 1mM, 2mM, 5mM, 10 mM, 20mM, 50mM, 100 mM. Other inhibitors of ATP producing enzymes include, but are not limited to, vanadate, disodium 4-nitrophenylphosphate (p-NPP), sodium pyrophosphate, and the like.
The inclusion of a cell lysing agent and/or ATP extracting agent in the additional component, in embodiments that quantitatively detect cell viability/intracellular ATP, reagents may be provided to lyse the cells and/or release ATP from the cells. Further, including nonionic detergents (e.g., triton-series detergents), cationic, anionic and zwitterionic detergents, bile salts, urea, thiourea, and any other cell membrane disrupting agent, including bacterial endolysins; or include any reagent that allows ATP to be extracted from the cells (e.g., CTAB).
Additional ingredients include one or more protein stabilizers, further suitable stabilizers include proteins (e.g., bovine serum albumin, gelatin, etc.), detergents (e.g., nonionic detergents such as threit), and the like.
Additional ingredients include substances known to enhance fluorescent signals and luminescence duration such as coenzyme A (CoA), thiol reagents (e.g., dithiothreitol) and metal ion chelators (e.g., EDTA and EGTA), protease inhibitors or salts (e.g., sodium chloride, potassium chloride, sodium sulfate, sodium bicarbonate, sodium dihydrogen phosphate, etc.).
The reagent composition and/or other components of the kit are contained in one or more containers. Further, the components of the reagent composition are contained in a single container, presented as a single component, or the components of the reagent composition are contained in multiple containers, presented as two or more components.
The reagent compositions and/or kits are provided in any kind of container in order to extend the shelf life of the different components. Further, the container may comprise any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramics, metals, or any other material commonly used for preserving reagents. Further, examples of suitable containers include kettles, bottles, envelopes, test tubes, vials, flasks, bottles, syringes and the like.
The test sample is any object containing or suspected of containing ATP or ATP analog, such as a cell lysate, intact cells, a biopsy, food, beverage, water, a swab rubbed over a surface such as an animal, plant, or inanimate object, or the like. Further, the cell or cell lysate may be from any organism, prokaryote or eukaryote. Further, eukaryotic cells may be derived from plants, animals, fungi, insects, etc., or cells cultured from these organisms.
In an eighth aspect, the present application provides an analysis system for quantitatively detecting ATP content in a sample, which adopts the following technical scheme:
an assay system for quantitatively detecting ATP content or concentration in a sample, comprising a reagent composition and a sample containing ATP.
The method for detecting the ATP content and the ATP concentration in the sample comprises the following steps:
(a) Adding a reagent composition to the sample;
(b) Quantifying the luminescence value of the sample;
(c) And comparing the luminescence with a standard quality control product, and calculating the ATP content or concentration in the sample.
In summary, the application has the following beneficial effects:
1. the firefly luciferase with high activity and high stability is obtained by carrying out random mutation on the firefly luciferase gene and screening the luminous intensity and half life.
2. The application adopts the kit composed of the firefly luciferase mutant and the firefly luciferin substrate to detect the ATP content, thereby improving the sensitivity and accuracy of detection.
Drawings
FIG. 1 is a graph comparing the activity of various luciferases (unmutated, S193I and L367I mutants) detected;
FIG. 2 is a graph showing the linear relationship between different luciferases (unmutated, S193I and L367I mutants) and ATP standard, and comparison of detection signal intensity;
FIG. 3 is a graph showing the relationship between luciferase (unmutated, S193I and L367I mutants) and HEK293 cell viability and comparison of detected signal intensities;
FIG. 4 is a graph comparing the detected signal intensities of different luciferases (unmutated, S193I and L367I mutants) for different cell lines;
FIG. 5 is a graph of the half-life of detection signals of different luciferases (unmutated, S193I and L367I mutants) versus ATP standard;
FIG. 6 is a graph comparing the half-life of detection signals of different luciferases (unmutated, S193I and L367I mutants) for cells.
Detailed Description
The examples are given solely for the purpose of illustration of the application and are not intended to limit the scope of the application. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the described embodiments, and the present application is not limited to the specific molecules, compositions, methods, or protocols described herein, as these may vary in accordance with routine experimentation and optimization.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Terminology
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, e.g., reference to "luciferase" refers to one or more luciferases known to those skilled in the art, analogs thereof, and the like.
As used herein, the term "enhancement" refers to an improvement over unmutated luciferase. For example, when used to describe a characteristic of a luciferase mutant (e.g., luminescence, signal stability, biocompatibility, protein stability (e.g., enzyme stability), or protein expression), "enhanced" refers to an improvement (e.g., 1%, 2%, 5%, 20%, 50%, 75%, 2%, 3%, 4%, 5%, 20%, 50%, 100%, 200%, 500%, 1000 or more fold) over a reference luciferase, e.g., a native/wild-type version of the luciferase SEQ ID NO:1. A mutated luciferase may exhibit one or more of "enhanced luminescence", "enhanced signal stability", "enhanced enzyme stability", "enhanced protein expression level", etc.
The term "signal stability" refers to the duration of a luminescent signal, e.g., measured by the half-life of the signal decaying over a period of time or the length of time the signal remains unchanged, and refers to the characteristic signal duration of an enzyme (e.g., luciferase). An enhancement of "signal stability enhancement" refers to an increase in the signal duration of a mutant luciferase as compared to an unmutated luciferase.
The term "amino acid" refers to natural amino acids, unnatural amino acids, and synthetic amino acid analogs, all in the D and L stereoisomers, unless otherwise indicated.
The term "sample" is used herein in its broadest sense. It comprises the following steps: a specimen, culture, lysate, etc. It includes a prepared solution or mixture, as well as biological and environmental samples. The biological sample may take the form of a fluid or a solid, and may be obtained from any suitable biological source (e.g., animals, including humans, microorganisms, etc.). Environmental samples include environmental materials such as surface matter, soil, plants, and water. The sample types of the present application, including but not limited to these examples.
Experimental materials, reagents and instruments
Bacteria: coli DH 5. Alpha. BL21 (DE 3).
And (3) cells: heLa cells, HEK293 cells, jurkat cells.
Reagent: bacterial culture reagents LB agarose powder, LB agar plates, antibiotics, cell culture reagents DMEM, RPMI1640, FBS, penicillin, and the like.
Consumable: white background opaque 96 well plates/384 well plates.
Instrument: ultraviolet visible spectrophotometers (spectromullab 752 s); bacteria shaking table (ZQZY-AF 8V known Chu instrument); ultrasonic cytoclasis apparatus (Biosafer 1000 China Sifei Co.); high-pressure cell disruptors (Shanghai Yonggang Biotechnology Co., ltd.); AKTApure protein purification system and matched Ni affinity chromatography pre-packed column and Superdex200 gel filtration chromatography pre-packed column (GE company in the United states); a Tanon gel imager (Shanghai Technical Co., ltd.); multifunctional microplate reader (INFINITE 200 Pro Tecan), etc.
Example 1 mutant construction and screening
The non-mutated firefly luciferase recombinant gene sequence is shown in U.S. Pat. No. 3,262B 2 (the coded protein is shown as SEQ ID NO: 1), the nucleotide sequence is artificially synthesized, and cloned into a vector pET-28a (+) to obtain a pET-28a (+) -Luc1 plasmid. A random mutant library was created by using the GeneMorph II random mutation kit (Agilent, cat. No. 200550) and the pET-28a (+) -Luc1 plasmid as templates. The mutant library was cloned into pET-28a (+) plasmid vector, the recombinant plasmid was transformed into BL21 (DE 3) competent cells, individual colonies of each mutant were picked up and cultured overnight in LB medium containing selection resistance, and the cultures were diluted into LB induction medium containing 0.5mM IPTG. The induced cultures were lysed and detected with detection reagents containing 1. Mu.M ATP, luciferin substrate. The RLU value for each mutant was calculated and the sample with the highest ratio was selected.
As a result, as shown in FIG. 1, both the S193I and L367I firefly luciferase mutants had higher activity. The signal intensities of the S193I and L367I firefly luciferase mutants were about 3.4-fold and 2.5-fold higher, respectively, than the unmutated firefly luciferase signal, with very significant differences. Finally, two mutation sites of S193I and L367I are determined, and mutant plasmids pET-28a (+) -Luc1-S193I and pET-28a (+) -Luc1-L367I are obtained.
SEQ ID NO:1
The amino acid sequence of firefly luciferase with no WT mutation is as follows:
MADKNILYGPEPFYPLEDGTAGEQMFDALSRYAAIPGCIALTNAHTKENVLYEEFLKLSCRLAESFKKYGLKQNDTIAVCSENSLQFFLPVIASLYLGIIVAPVNDKYIERELIHSLGIVKPRIVFCSKNTFQKVLNVKSKLKSIETIIILDLNEDLGGYQCLNNFISQNSDSNLDVKKFKPYSFNRDDQVASIMFSSGTTGLPKGVMLTHKNIVARFSIAKDPTFGNAINPTSAILTVIPFHHGFGMMTTLGYFTCGFRVVLMHTFEEKLFLQSLQDYKVESTLLVPTLMAFLAKSALVEKYDLSHLKEIASGGAPLSKEIGEMVKKRFKLNFVRQGYGLTETTSAVLITPKGDAKPGSTGKIVPLHAVKVVDPTTGKILGPNEPGELYFKGPMIMKGYYNNEEATKAIIDNDGWLRSGDIAYYDNDGHFYIVDRLKSLIKYKGYQVAPAEIEGILLQHPYIVDAGVTGIPDEAAGELPAAGVVVQTGKYLNEQIVQDYVASQVSTAKWLRGGVKFLDEIPKGSTGKIDRKVLRQMLEKHTNG
SEQ ID NO:2
the amino acid sequence of the MT-S193I firefly luciferase mutant is as follows:
MADKNILYGPEPFYPLEDGTAGEQMFDALSRYAAIPGCIALTNAHTKENVLYEEFLKLSCRLAESFKKYGLKQNDTIAVCSENSLQFFLPVIASLYLGIIVAPVNDKYIERELIHSLGIVKPRIVFCSKNTFQKVLNVKSKLKSIETIIILDLNEDLGGYQCLNNFISQNSDSNLDVKKFKPYSFNRDDQVAIIMFSSGTTGLPKGVMLTHKNIVARFSIAKDPTFGNAINPTSAILTVIPFHHGFGMMTTLGYFTCGFRVVLMHTFEEKLFLQSLQDYKVESTLLVPTLMAFLAKSALVEKYDLSHLKEIASGGAPLSKEIGEMVKKRFKLNFVRQGYGLTETTSAVLITPKGDAKPGSTGKIVPLHAVKVVDPTTGKILGPNEPGELYFKGPMIMKGYYNNEEATKAIIDNDGWLRSGDIAYYDNDGHFYIVDRLKSLIKYKGYQVAPAEIEGILLQHPYIVDAGVTGIPDEAAGELPAAGVVVQTGKYLNEQIVQDYVASQVSTAKWLRGGVKFLDEIPKGSTGKIDRKVLRQMLEKHTNG
SEQ ID NO:3
the amino acid sequence of the MT-L367I firefly luciferase mutant is as follows:
MADKNILYGPEPFYPLEDGTAGEQMFDALSRYAAIPGCIALTNAHTKENVLYEEFLKLSCRLAESFKKYGLKQNDTIAVCSENSLQFFLPVIASLYLGIIVAPVNDKYIERELIHSLGIVKPRIVFCSKNTFQKVLNVKSKLKSIETIIILDLNEDLGGYQCLNNFISQNSDSNLDVKKFKPYSFNRDDQVASIMFSSGTTGLPKGVMLTHKNIVARFSIAKDPTFGNAINPTSAILTVIPFHHGFGMMTTLGYFTCGFRVVLMHTFEEKLFLQSLQDYKVESTLLVPTLMAFLAKSALVEKYDLSHLKEIASGGAPLSKEIGEMVKKRFKLNFVRQGYGLTETTSAVLITPKGDAKPGSTGKIVPIHAVKVVDPTTGKILGPNEPGELYFKGPMIMKGYYNNEEATKAIIDNDGWLRSGDIAYYDNDGHFYIVDRLKSLIKYKGYQVAPAEIEGILLQHPYIVDAGVTGIPDEAAGELPAAGVVVQTGKYLNEQIVQDYVASQVSTAKWLRGGVKFLDEIPKGSTGKIDRKVLRQMLEKHTNG
EXAMPLE 2 mutant solubility assay
The template plasmid pET-28a (+) -Luc1 and 2 mutant plasmids were transformed into BL21 (DE 3) competent cells and cultured overnight at 37℃on LB plates containing antibiotics. The following day, the single clones were picked up and inoculated into 3ml LB (K) + Resistance) in the culture solution, shaking culture is carried out for 8-12 hours at 37 ℃ at 200rpm/min, and the strain is reserved for the next day of induced expression. The bacterial liquid was added to 3ml of LB liquid medium by pipetting, incubated at 37℃for 2 hours in a incubator at 200rpm/min, and the absorbance of the bacterial liquid was measured by using a spectrophotometer, and when the OD600nm value was about 0.8, the bacterial liquid was taken out. Standing the bacterial liquid at 16 ℃ for 30min, adding IPTG with the final concentration of 0.5mM, culturing for 24h at 180rpm/min at 16 ℃, and simultaneously establishing a non-induction group; after the induction was completed, the cells were collected using a centrifuge at 4℃and resuspended using 500. Mu.L of PBS. The cells were sonicated in an ice bath at 13,000Xrpm at 4℃for 5min and the supernatant was collected. SDS-PAGE electrophoresis and Coomassie brilliant blue staining of each enzyme supernatant were performed, and it was observed that the target proteins were expressed in a soluble form. The protein purity can reach more than 99% through two-step purification of Ni affinity chromatography purification column and Superdex200 gel filtration chromatography pre-packed column.
EXAMPLE 3 expression and purification of preferred firefly luciferin mutants
BL21 (DE 3) strain of firefly luciferase mutant (mutant plasmids pET-28a (+) -Luc1-S193I and pET-28a (+) -Luc 1-L367I) and strain containing unmutated plasmid (pET-28 a (+) -Luc 1-containing plasmid) were grown on K + Induction culture was performed in resistant LB liquid medium, and specific conditions are described in example 2. After the induction is finished, the thalli are collected by a centrifugal machine at the temperature of 4 ℃ and at the speed of 8000 rpm/min: after the lysate (50mM Tris,500mM NaCl,5% glycerol, pH 7.5) was fully resuspended at a ratio of 1:10, the cells were broken up in a high-pressure breaker, the broken supernatant was collected by centrifugation at high speed at 20000rpm/min at 4℃and purified coarsely by Ni affinity chromatography purification columns, then fine purified by Superdex200 gel filtration chromatography pre-packed columns, and the stock was replaced with (50mM Tris,150mM NaCl,5% glycerol, pH 7.5) and detected by SDS-PAGE and Coomassie brilliant blue staining. The purified luciferase was assayed for concentration using Pierce ™ BCA protein quantification kit (thermosusher, 23225) and stored in aliquots at-80 ℃.
Example 4 firefly luciferase mutant for detection of ATP Standard Activity Curve
The effect of firefly luciferase mutant use was determined by titration of ATP.
To confirm that the mutant firefly luciferases described herein were used for detection of ATP standard, detection reagents (40. Mu.g/ml firefly luciferase, 50mM Hepes,pH 7.0, 300 mM NaCl,1mM EDTA, 1%Triton X-100, 20mM MgSO were prepared 4 0.1% gelatin and 5% glycerol, 1mM D-Luciferin).
Sample treatment: preparation of ATP standard with sterile, enzyme-free water to 1. Mu.M ATP (100. Mu.l containing 10) -10 Molar ATP), in a 2-fold dilution ratio (1. Mu.M to 10 nM, 100. Mu.l containing 10 -10 To 10 -12 Molar ATP). 100 μl of ATP standard dilution was added to each well of a white background opaque 96 well plate, an equal volume of detection reagent (containing luciferase substrate and purified firefly luciferase) was added, the contents were mixed using an orbital shaker for 2min, and the plate was incubated at room temperatureIncubating for 10min to stabilize the luminescence signal. The luminescence signal is recorded.
The results show that both the S193I and L367I mutants exhibited stronger luminescence values and detection sensitivities compared to the unmutated firefly luciferase. The unmutated, S193I and L367I mutant firefly luciferases all show good linear relation to the detection luminescence values of the ATP standard. Wherein the S193I firefly luciferase mutant exhibits the strongest detection effect, and the RLU value is 3.4 times that of the unmutated firefly luciferase signal. The L367I firefly luciferase mutant was 2.3-fold the unmutated firefly luciferase signal (FIG. 2).
Example 5 firefly luciferase mutant for detection of cell Activity Curve
The effect of use of firefly luciferase mutants was further determined by titration tests on HEK293 cells.
To confirm that the mutant firefly luciferases described herein were used for detection of ATP standard, detection reagents (40. Mu.g/ml firefly luciferase, 50mM Hepes,pH 7.0, 300 mM NaCl,1mM EDTA, 1%Triton X-100, 20mM MgSO were prepared 4 0.1% gelatin and 5% glycerol, 1mM D-Luciferin). Sample treatment: HEK293 cells were subjected to 2-fold gradient dilution in 96-well plates using DMEM medium (10% fbs) and left to stand at room temperature for about 30min. An equal volume of detection reagent (containing luciferase substrate and purified firefly luciferase) was added to each well of cell culture medium, the contents were mixed for 2min using an orbital shaker, and the plates were incubated at room temperature for 10min to stabilize the luminescence signal. The luminescence signal is recorded.
The results show that both the S193I and L367I mutants exhibited stronger luminescence values and detection sensitivities compared to the unmutated firefly luciferase. The unmutated, S193I and L367I mutant firefly luciferases all show good linear relation to the detection luminescence values of HEK293 cells. Wherein the S193I firefly luciferase mutant exhibits the strongest detection effect, and the RLU value is 3.6 times that of the unmutated firefly luciferase signal. The L367I firefly luciferase mutant was 3.0 times the unmutated firefly luciferase signal (FIG. 3).
Example 6 firefly luciferase mutant for viability assay of different cells
To confirm that the mutant firefly luciferases described herein were used for different cell line assays, assay reagents (80. Mu.g/ml firefly luciferase, 50mM MES,pH 6.0, 100 mM NaCl,1mM EDTA,2%Triton X-100, 40 mM MgSO were prepared 4 0.5mM D-Luciferin). Samples were selected from 3 common cell lines HeLa, HEK293 and Jurkat cells. White-bottomed opaque 96-well plates were seeded at a density of 2 ten thousand per well, equilibrated for 30min at room temperature, added with an equal volume of detection reagent (luciferase-containing substrate and purified firefly luciferase) per well of cell culture medium, the contents were mixed for 2min using an orbital shaker, and the plates were incubated at room temperature for 10min to stabilize the luminescence signal. The luminescence signal is recorded.
The results show that, as the detection method is a detection means based on ATP content, for the activity detection of HEK293, heLa and Jurkat cells, unmutated S193I and L367I mutant firefly luciferases all show different detection effects due to cell line differences (figure 4). Wherein, the S193I firefly luciferase mutant has stronger detection effect, and the RLU value is 4.2 times of that of the unmutated firefly luciferase signal. The L367I firefly luciferase mutant was 3.3 times the unmutated firefly luciferase signal.
Example 7 firefly luciferase mutant luminescence Signal stability assay with respect to ATP Standard
To determine the luminescence signal stability of the mutant firefly luciferases described herein, detection reagents (100. Mu.g/ml firefly luciferase, 20mM citrate,pH 6.0, 150mM NaCl,2mM EDTA,2%Triton X-100, 50mM MgSO were prepared 4 0.2% gelatin, 5% glycerol, 2mM D-Luciferin) to determine the stability of the luminescent signal of a 1. Mu.M ATP standard.
Sample treatment: ATP standard was prepared in 1. Mu.M ATP dilution (100. Mu.l containing 10) using enzyme-free sterile water -10 Molar ATP), 100 μl of ATP standard dilution was added to each well of a white-bottomed opaque 96-well plate, and an equal volume of detection reagent (containing luciferin) was addedEnzyme substrate and purified firefly luciferase), the contents were mixed for 2min using an orbital shaker, and the plates were incubated at room temperature for 10min to stabilize the luminescence signal. The luminescence signal was recorded every 10 min.
The results show that the signal half-life of the non-mutant, S193I and L367I mutant firefly luciferases exceeded 5 hours in the detection of 1. Mu.M ATP standard. The luminescence signal of the S193I and L367I mutant firefly luciferases could still reach more than 90% of the initial luminescence value within 60min (FIG. 5).
Example 8 firefly luciferase mutant stability assay with respect to luminescent signal from cell samples
To determine the luminescence signal stability of the mutant firefly luciferases described herein, detection reagents (100 μg/ml firefly luciferase, 20mM citrate, pH 6.0, 150mM NaCl,2mM EDTA,2%Triton X-100, 50mM MgSO4,0.2% gelatin, 5% glycerol, 2mM D-Luciferin) were prepared for use in determining the luminescence signal stability of HEK293 cells.
HEK293 cells were seeded at a density of 2 ten thousand per well in white-bottomed opaque 96-well plates, equilibrated for 30min at room temperature, detection reagents (containing luciferase substrate and pure firefly luciferase) were added in an equal volume to the cell culture medium per well, the contents were mixed for 2min using an orbital shaker, and the plates were incubated at room temperature for 10min to stabilize the luminescence signal. The luminescence signal was recorded every 10 min.
The results show that in the detection of HEK293 cell viability, both the signal half-lives of the S193I and L367I mutant firefly luciferases exceeded 5 hours, and the non-mutant half-life was only about 4 hours. The luminescence signal of the S193I and L367I mutant firefly luciferases can still reach more than 85% of the initial luminescence value within 60min, and the activity of the non-mutant is reduced by 25% and is only 75% of the initial luminescence signal (figure 6).
Claims (10)
1. A firefly luciferase mutant characterized in that: the amino acid sequence of firefly luciferase shown in SEQ ID NO. 1 is mutated at the 193 rd and/or 367 th amino acids, the 193 rd serine Ser S is mutated into isoleucine Ile I or the 367 th serine Leu L is mutated into isoleucine Ile I, and the amino acid sequence is shown in SEQ ID NO. 2 or SEQ ID NO. 3 respectively.
2. A protein, characterized in that: is any one of the following (a 1) to (a 3):
(a1) An enzyme mutated at the position 193 and the position 367 of the amino acid sequence shown in SEQ ID NO. 1 of claim 1;
(a2) An enzyme derived from (a 1) having the function/activity of the enzyme of (a 1) and formed by substitution and/or deletion and/or addition of an amino acid residue at amino acid residues 193 and/or 367 in the amino acid sequence of the enzyme of (a 1);
(a3) A polypeptide formed by connecting the N end or/and the C end of the mutated amino acid sequence of the (a 1) or the (a 2) with a tag sequence or an enzyme cutting site sequence, and a fusion protein.
3. A nucleic acid molecule characterized in that: comprising a nucleic acid molecule as in (b 1) or (b 2) below:
(b1) The nucleotide sequence is a DNA molecule encoding the protein of claim 2;
(b2) A DNA molecule having at least 95% identity to the nucleotide sequence of (b 1) and encoding the protein of claim 2.
4. A recombinant vector, characterized in that: a nucleic acid molecule comprising the nucleic acid molecule of claim 3.
5. A recombinant bacterium, characterized in that: comprising the recombinant vector or genome of claim 4 having incorporated therein the nucleic acid molecule of claim 3.
6. A method for preparing firefly luciferase mutant according to claim 1, wherein: the artificial synthesized SEQ ID NO. 1 nucleotide sequence is taken as a template, a random mutant library is established through a random mutation kit, the mutant library is cloned into a prokaryotic expression vector, BL21 (DE 3) competent cells are further transferred to induce the expression of the mutant library, a culture is cracked and detected, the RLU value of each mutant is calculated, and a sample with the highest ratio is selected, so that the mutant luciferase is obtained.
7. A reagent composition characterized by: a firefly luciferase mutant comprising the firefly luciferase mutant of claim 1 and a firefly luciferin substrate.
8. The reagent composition of claim 7, wherein: magnesium ions are also included.
9. The reagent composition of claim 7, wherein: further comprises an additional component comprising at least one of a buffer, a salt, a protease inhibitor, a cell lysing agent, a detergent, an ATPase inhibitor, an ATP producing enzyme inhibitor, an ATP extracting agent, conA, a thiol reagent, or a metal ion chelating agent.
10. An assay system for quantitatively detecting ATP content or concentration in a sample comprising the reagent composition of claim 7 and a sample comprising ATP.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010081908A (en) * | 2008-10-02 | 2010-04-15 | Hitachi Ltd | Mutated firefly luciferase |
| CN102597231A (en) * | 2009-11-10 | 2012-07-18 | 龟甲万株式会社 | Firefly luciferase and gene thereof, and process for production of firefly luciferase |
| JP2012235756A (en) * | 2011-05-13 | 2012-12-06 | Olympus Corp | Luciferase derived from firefly |
| CN113061591A (en) * | 2021-03-31 | 2021-07-02 | 上海碧云天生物技术有限公司 | Novel firefly luciferase mutant, preparation method and application thereof |
| WO2022076903A1 (en) * | 2020-10-09 | 2022-04-14 | Atea Pharmaceuticals, Inc. | Niran interfering drugs for sars-cov-2 mutant therapy |
| CN114350627A (en) * | 2021-12-16 | 2022-04-15 | 大连博格林生物科技有限公司 | Firefly luciferase mutant and preparation method thereof |
| CN116287106A (en) * | 2022-12-30 | 2023-06-23 | 上海碧云天生物技术有限公司 | Method and reagent for enhancing firefly luciferase ATP bioluminescence detection performance |
| CN116555204A (en) * | 2023-07-04 | 2023-08-08 | 苏州驾玉生物医药有限公司 | Mutant luciferase with improved performance and application thereof |
-
2023
- 2023-08-22 CN CN202311058580.5A patent/CN116814568B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010081908A (en) * | 2008-10-02 | 2010-04-15 | Hitachi Ltd | Mutated firefly luciferase |
| CN102597231A (en) * | 2009-11-10 | 2012-07-18 | 龟甲万株式会社 | Firefly luciferase and gene thereof, and process for production of firefly luciferase |
| JP2012235756A (en) * | 2011-05-13 | 2012-12-06 | Olympus Corp | Luciferase derived from firefly |
| WO2022076903A1 (en) * | 2020-10-09 | 2022-04-14 | Atea Pharmaceuticals, Inc. | Niran interfering drugs for sars-cov-2 mutant therapy |
| CN113061591A (en) * | 2021-03-31 | 2021-07-02 | 上海碧云天生物技术有限公司 | Novel firefly luciferase mutant, preparation method and application thereof |
| CN114350627A (en) * | 2021-12-16 | 2022-04-15 | 大连博格林生物科技有限公司 | Firefly luciferase mutant and preparation method thereof |
| CN116287106A (en) * | 2022-12-30 | 2023-06-23 | 上海碧云天生物技术有限公司 | Method and reagent for enhancing firefly luciferase ATP bioluminescence detection performance |
| CN116555204A (en) * | 2023-07-04 | 2023-08-08 | 苏州驾玉生物医药有限公司 | Mutant luciferase with improved performance and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| CALABRETTA MM等: "A LuciferaseMutant with Improved Brightness and Stability for Whole-Cell Bioluminescent Biosensors and In Vitro Biosensing", 《BIOSENSORS (BASEL)》, vol. 12, no. 9, pages 742 * |
| FUJII H等: "Increase in bioluminescence intensity of firefly luciferase using genetic modification", 《ANALYTICAL BIOCHEMISTRY》, vol. 366, no. 2, pages 131 - 136, XP022126352, DOI: 10.1016/j.ab.2007.04.018 * |
| 刘琳等: "基于荧光素酶生物发光检测方法的研究及其应用进展", 《中国药科大学学报》 * |
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