Luciferase mutant and use thereof

Abstract

The application discloses a luciferase mutant, wherein the amino acid sequence is shown as SEQ ID NO. 1, and the polynucleotide sequence is shown as SEQ ID NO. 2. The catalytic activity of the luciferase mutant is 3 times higher than that of wild type, the luciferase mutant can be prepared through industrialization, and the luciferase mutant can be used for preparing a luciferase detection kit and used as a'reporter protein' or'reporter gene' for biological research.

Description

TECHNICAL FIELD

[0001] The application discloses a protein mutant and belongs to the technical field of polypeptides. BACKGROUND

[0002] Luciferase is a general term for enzymes that can produce bioluminescence in nature, among which the most representative is a luciferase in fireflies with the scientific name of Photinus pyralis. Photinus pyrali The catalytic reaction of luciferase is as follows: in the presence of magnesium ions, luciferin and ATP are first catalyzed to generate luciferin-AMP and pyrophosphate, and then luciferin-AMP and oxygen are catalyzed to generate oxidized luciferin, AMP and carbon dioxide, while releasing fluorescence. When the enzyme content is consistent and the substrate is excessive in the luciferase reaction system, the light intensity is proportional to the luciferase activity. The luciferase-catalyzed reaction emits yellow-green light at a pH value of 7.5-8.5, and the wavelength of the emitted light is 562-570 nm. The emitted photons can be detected by light-sensitive elements such as a fluorescence detector or an improved optical microscope, which makes it possible to observe the progress of various life activities.

[0003] Luciferase can be synthesized by genetic engineering methods and used in various detection experiments. Luciferase can be used as a "reporter protein" in molecular biology research, for example, to detect the transcription of a specific promoter in cells transfected with luciferase or to detect ATP levels in cells; this technology is called reporter gene detection or luciferase assay. A reporter gene or reporter group is a chemical group, protein or enzyme that can be easily detected by experimental instruments and the genes encoding them, so it is widely used in experiments. For example, luciferase gene used in luciferase reporter gene experiment, fluorescence reporter group in qPCR. Reporter genes are widely used in gene expression regulation, signal transduction, promoter analysis, receptor function identification, gene therapy and drug screening. Luciferase is also a heat-sensitive protein and is often used to study the protective ability of heat shock proteins in the process of protein thermal denaturation.

[0004] Luciferase has become an ideal reporter protein due to its excellent sensitivity, convenience of use, and quantitative detection. Therefore, there is great technical application and market demand for new luciferase and its applications.

[0005] The purpose of the present application is to provide a luciferase mutant with higher enzyme activity. SUMMARY

[0006] Based on the above-mentioned purpose of the application, the present application first provides a luciferase mutant, wherein the amino acid sequence of the luciferase mutant is shown as SEQ ID NO. 1.

[0007] Secondly, the present application provides a polynucleotide encoding the luciferase mutant. According to the common knowledge of those skilled in the art, any polynucleotide capable of encoding the luciferase mutant with the amino acid sequence shown in SEQ ID NO. 1 belongs to the polynucleotide of the present application.

[0008] In a preferred embodiment, the sequence of the polynucleotide is shown in SEQ ID NO. 2.

[0009] Thirdly, the present application provides an expression vector containing the polynucleotide. As known by those skilled in the art, the luciferase mutant of the present application can be prepared by genetic engineering methods, and the conventional expression vectors, such as prokaryotic expression vectors or eukaryotic expression vectors, can be used for the industrial production of the luciferase mutant.

[0010] Fourthly, the present application provides a host cell containing the expression vector. As known by those skilled in the art, the expression vector can be introduced into host cells, such as prokaryotic host cells like Escherichia coli or eukaryotic host cells like yeast, to achieve the industrial production of the luciferase mutant.

[0011] Fifthly, the present application provides a method for screening luciferase mutants, which comprises the following steps:

[0012] (1) introducing a first vector containing an nCas9 coding gene and a cytosine deaminase coding gene into a yeast strain, and integrating the nCas9 coding gene and the cytosine deaminase coding gene into the GS115 genome by homologous recombination to obtain a recombinant yeast strain containing the nCas9 coding gene and the cytosine deaminase coding gene in the genome;

[0013] (2) introducing a second vector containing a luciferase coding gene into the recombinant yeast strain obtained in step (1), and integrating the luciferase coding gene into the genome of the recombinant strain by homologous recombination to screen a recombinant yeast strain with a single copy of the luciferase coding gene, wherein the second vector carries an inducible promoter element;

[0014] (3) introducing a gRNA plasmid library targeting the luciferase coding gene into the recombinant yeast strain obtained in step (2) to mutate the luciferase coding gene, wherein the gRNA plasmid library contains 51 sequences targeting different segments of the luciferase coding gene, as shown in SEQ ID NO. 3-53, and the gRNA is constructed on a third vector;

[0015] (4) inducing the yeast strain obtained in step (3) to express the luciferase gene;

[0016] (5) Screening luciferase mutants according to the enzyme activity of luciferase expression product of step (4).

[0017] In a preferred technical solution, the yeast strain is GS115.

[0018] In a specific embodiment of the present application, the first vector is pGAP, and the first vector containing the nCas9 coding gene and cytosine deaminase coding gene of step (1) is named "pGAP-nCas9-(GGGGS) 10 -PmCDA1" in the present application, and the recombinant yeast strain containing the nCas9 coding gene and cytosine deaminase coding gene is named "GS115-nCas9 -PmCDA1" in the present application.

[0019] In a specific embodiment of the present application, the second vector is pPIC9K, which contains the AOX1 promoter and can strictly control the transcription and expression of foreign coding genes by methanol. The second vector containing the luciferase coding gene of step (2) is named "pPIC9K-Luc" in the present application, and the recombinant yeast strain with a single copy of the luciferase coding gene is named "GS115-nCas9 -PmCDA1-Luc" in the present application.

[0020] In a specific embodiment of the present application, the third vector is pTEF, which contains the Sh ble expression frame and can be used for screening of recombinant strains. The third vector containing 51 target luciferase coding gene different segment sequences such as SEQ ID NO. 3-53, gRNA of step (3) is named "pTEF-gRNA" in the present application.

[0021] Sixth, the application of the luciferase mutant in the preparation of luciferase detection kit. The luciferase detection method of the present application produces fluorescence intensity through the action of luciferase and substrate such as ATP, so as to detect the substrate and other target substances associated therewith.

[0022] Seventh, the present application provides the application of the above-mentioned luciferase mutant as a reporter protein. By associating luciferase with target molecules (high sensitivity and rapid luminescence reaction), the luminescence signal thereof can be used to evaluate the activity of compounds in a compound library or the effect of inhibitors to realize high-throughput screening. Luciferase can also be used to construct biosensors for detecting the presence and concentration changes of specific substances. By combining luciferase with appropriate sensors, quantitative or localization detection of target molecules in biological samples can be realized.

[0023] Finally, the present application provides the polynucleotide encoding the luciferase mutant as described above for use as a luciferase reporter gene, which further comprises a promoter and / or a polynucleotide encoding a gene. For example, luciferase is often used as a bioluminescent reporter gene for studying biological processes such as gene expression, transcriptional regulation, and protein-protein interactions. By linking the luciferase gene to the gene or promoter of interest, the expression level or activity of the gene or promoter can be monitored in real time. In addition, luciferase can be used as a marker for biomolecular imaging. By introducing the luciferase gene into target cells or tissues, when a specific substrate (such as luciferin) is present, luciferase will catalyze a reaction that emits visible light signals, enabling real-time observation of specific locations or processes, such as luciferase-labeled tumor cells showing changes in tumor size in vivo.

[0024] The present application uses Pichia pastoris as a host to mutate luciferase sequences using CRISPR-mediated base editors and perform high-throughput screening, obtaining a mutant with high sensitivity to ATP. The luciferase mutant has higher enzyme catalytic activity, which is 3 times higher than the wild-type luciferase. The luciferase mutant can be prepared through industrialization. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 . pPIC9K-Luc plasmid map;

[0026] Figure 2 . Distribution diagram of 51 gRNAs in pPIC9K-Luc;

[0027] Figure 3 . Comparison chart of fluorescence intensity of 9 mutant strains and wild-type luciferase;

[0028] Figure 4 . Comparison chart of fluorescence intensity of 4 mutant strains and wild-type luciferase;

[0029] Figure 5 . Sequence alignment chart of Luc-3 mutant and wild-type luciferase;

[0030] Figure 6 . Comparison chart of fluorescence intensity of Luc-3 mutant and wild-type luciferase;

[0031] Figure 7 . Comparison chart of fluorescence intensity of Luc-3 mutant and control group. DETAILED DESCRIPTION

[0032] The present application will be further described below in conjunction with specific examples, and the advantages and characteristics of the present application will become more apparent as the description proceeds. However, these examples are only exemplary and do not constitute any limitation on the protection scope defined by the claims of the present application.

[0033] The experimental reagents involved in the present application are: proteose peptone, yeast extract (Oxoid Company), agar powder (Novon Company), glucose, sodium chloride, potassium dihydrogen phosphate (Xilong Scientific Co., Ltd.), glycerol, sodium hydroxide (Jiangsu Qiangsheng Functional Chemical Co., Ltd.), methanol (National Pharmaceutical Group Chemical Reagent Co., Ltd.), trihydrate potassium phosphate (Chengdu Kolon Chemical Co., Ltd.), plasmid extraction kit (Qiagen Company), SDS-PAGE gel preparation kit (Jinsirui Biological Technology Co., Ltd.), gel recovery kit (Omega Company), YNB (Yeast Nitrogen Base w / o AminnoAcids), sorbitol (Sigma Company), XhoI, EcoRI, SacI, AflII restriction endonuclease and Quick Ligase enzyme (New England Biolabs Company), 2*Phanta Flash Master Mix, ClonExpress Multis OneStep Cloning Kit (Vazyme Company), KOD enzyme (TOYOBO Company), Zeocin (Z + , bleomycin, Invitrogen Company), G418 (Thermo Fisher Scientific Company), TOP10 competent (Tiangen Biotech Co., Ltd.), pGAP vector (Invitrogen Company), pPIC9K vector (Invitrogen Company), pTEF vector (Addgene Company), luciferase detection kit (Shandong Yuntang Intelligent Technology Co., Ltd.).

[0034] The culture media involved in the present application are: YPD culture medium (1% yeast extract, 2% proteose peptone, 2% glucose), YPD S Z + culture medium (1% yeast extract, 2% proteose peptone, 2% glucose, 0.1% Z + resistance, 1 M sorbitol), MD culture medium (1.34% YNB, 2% glucose), YPD G418 culture medium (1% yeast extract, 2% proteose peptone, 2% glucose, 2% (V / V) G418), LB culture medium (0.5% yeast extract, 1% proteose peptone, 1% sodium chloride), LLB culture medium (0.5% yeast extract, 1% proteose peptone, 0.5% sodium chloride), MGY culture medium (1% yeast extract, 2% proteose peptone, 1.34% YNB, 1 M potassium phosphate and 2% glycerol), MMY culture medium (1% yeast extract, 2% proteose peptone, 1.34% YNB, 1 M potassium phosphate, 0.5% methanol), and all culture media are configured into solid plates with 2% agar powder.

[0035] The solutions involved in the present application are: 10x glucose (200 g glucose is made up to 1 L, sterilized at 115°C for 15 min), 10x glycerol (100 g glycerol is made up to 1 L, sterilized by filtration with a 0.22 μm filter), 10x YNB (34 g YNB and 100 g ammonium sulfate are made up to 1 L, sterilized by filtration with a 0.22 μm filter), 1 M sorbitol (182.17 g sorbitol is made up to 1 L, sterilized by filtration with a 0.22 μm filter), 1 M phosphate buffer (118.12 g potassium dihydrogen phosphate and 30.12 g dihydrogen phosphate, pH value is adjusted to 6.0 ± 0.1 with potassium hydroxide).

[0036] Example 1. Screening of luciferase mutants

[0037] 1. Construction of monoclonal stably expressing luciferase

[0038] (1) Construction of recombinant strain GS115-nCas9-PmCDA1

[0039] The gene encoding cytosine deaminase PmCDA1 (GenBank: ABO15149.1) is fused to the C-terminal of Cas9 (GenBank: KT031982.1) point (D10A) mutant (nCas9), and the two are connected by a linker (GGGGS) 10 linker, which is synthesized by the company nCas9-(GGGGS) 10 -PmCDA1 fragment, the recombinant plasmid pGAP-nCas9-(GGGGS) 10 -PmCDA1, which contains a HIS4 expression frame and can be used for yeast transformation screening. The linearized recombinant plasmid obtained by Nhe I restriction enzyme is electroporated into GS115 yeast bacteria, and the electroporation parameters are 2000 Voltage, 25 Capacitance (μF), 200 Resistance (Ω), 20 Cuvette (mm), so that it is integrated into the GS115 genome by homologous recombination, and the positive clones are screened using MD plates, and identified by colony PCR with primers GAP1-F / CYC1-R (5'-CGTCGCTGGCAATAATAGCG-3' / 5'-CCTTCCTTTTCGGTTAGAGC-3'), and the GS115-nCas9-PmCDA1 recombinant strain is obtained after sequencing confirmation.

[0040] (2) Construction of recombinant plasmid pPIC9K-Luc

[0041] Synthesized by GenScript (Nanjing) Co., Ltd. Allobacillus saliphilus The nucleotide sequence of luciferase from P. pastoris (GenBank: AOR57259.1) was used to construct recombinant plasmid pPIC9K-Luc (see plasmid map in Figure 1 ), which contains AOX1 promoter and can be strictly controlled by methanol to induce transcription. The recombinant plasmid was transformed into TOP10 competent cells by heat shock method and screened by LB solid plate containing ampicillin to obtain a single colony. The specific operation was as follows: 5 μg of synthetic plasmid containing Luc and pPIC9K vector were respectively digested with 2 μL Xho I / Eco R I restriction enzymes for 3 h, and then recovered to 20 μL ultrapure water. The recovered target fragment and linearized vector were connected with Quick Ligase enzyme for 30 min, and then transformed into TOP10 competent cells by heat shock at 42°C. Then 900 μL of LB was added and placed in a 37°C shaker at 220 rpm for 45 min. The bacterial solution was then spread on LB A + screening plate and incubated at 37°C for 14-16 h to obtain a single colony. Ten single colonies were randomly selected for colony PCR and sequencing, and a single colony containing the correct recombinant plasmid was obtained. One correct single colony was selected for culture, and about 25 μg of recombinant plasmid was obtained using QIAGEN plasmid extraction kit.

[0042] (3) Screening of single-copy positive strain GS115-nCas9-PmCDA1-Luc

[0043] Take 50 μL of GS115-nCas9-PmCDA1 yeast solution and add 5 mL of YPD liquid medium, shake at 30°C until over-cultured, add 2 mL of over-cultured yeast to 200 mL of YPD liquid medium, and incubate at 30°C constant temperature shaker for 8 h until the OD600 value is slightly greater than 1.0. Centrifuge the bacteria, wash twice with sterile water, wash once with 1 M sorbitol, resuspend the yeast in 1 M sorbitol, and dilute to 1.5 mL (ice bath). Complete the preparation of yeast competent cells. Linearize the recombinant plasmid pPIC9K-Luc with restriction enzyme Sac I, and then integrate it into the genome of the competent cells by electroporation. The electroporation parameters are: 2000 Voltage, 25 Capacitance (μF), 200 Resistance (Ω), 20 Cuvette (mm). Screen with G418 plate, randomly select 5 single colonies for colony PCR, culture the single colony containing the target sequence for 24 h, induce with 0.5% methanol, select a single colony that can successfully express luciferase and emit fluorescence with the substrate luciferin, shake the bacteria, preserve the bacteria, and keep it as a strain.

[0044] 2. Construction of gRNA plasmid library

[0045] According to the nucleotide sequence of luciferase, 51 target gRNAs (SEQ ID NO. 3-53, see Figure 2 ) were designed and integrated into pTEF vector by homologous recombination to obtain recombinant plasmid pTEF-gRNA. The vector contains Sh ble expression frame, which can be selected by Zeocin (Z+) for screening of recombinant strains. The recombinant plasmid was transformed into TOP10 competent cells by heat shock method and selected by 0.1% Z + resistant LLB solid plate to obtain single colonies. The specific operation is as follows: add homologous fragments (5'-AGGACGAAACGAGTAAGCTCGTCTCAGATC-3' / 5'- ATTTTAACTTGCTATTTCTAGCTCTAAAAC-3') to the two primers of the 51 gRNAs to form gRNA primers, and then use PCR amplification to obtain the target fragment containing gRNA. Take 5 μg pTEF vector and add 4 μL restriction endonuclease Afl II, incubate at 37°C for 2 h to complete the linearization of the vector, and then recover the gel into 30 μL ultrapure water. Take 1 μL amplification product and 1 μL linearized vector respectively and use Exnase Multis for homologous recombination. Transform into TOP10 competent cells at 42°C, then add 900 μL LB and place in a 37°C shaker at 220 rpm for 45 min, then spread the bacterial solution on LLB Zeocin (Z + ) resistant plate and incubate at 37°C for 14-16 h to obtain single colonies. Randomly select 10 single colonies for colony PCR and sequencing to obtain single colonies containing correct recombinant plasmids.

[0046] Culture the correctly constructed single colonies and use QIAGEN plasmid extraction kit to obtain recombinant plasmids. Take 200 ng of each recombinant gRNA plasmid to form a gRNA plasmid library.

[0047] 3. Construction and screening of mutant library

[0048] Take 1 μg of plasmid in the gRNA library and electroporate into GS115-nCas9-Luc competent cells to use YPDS Z + plate for screening to obtain 1632 single colonies. First, use MGY medium to culture all single colonies for 24 h, then replace with MMY medium for induction for 24 h, and add 0.5% methanol every 24 h for a total of 72 h of induction and expression.

[0049] The 10 μL induction supernatant and 90 μL reaction system with substrate luciferin were added to a 96-well plate, and then the plate was placed in a Glomax instrument after uniform blowing. The integration time was set to 4 s, and the chemiluminescence intensity was detected. The luciferase reaction system was derived from the literature (doi: 10.7546 / ijba.2019.23.3.000720) and was simplified, and the details are shown in Table 1.

[0050] Table 1. Luciferase reaction system

[0051]

[0052] Chemiluminescence screening was performed on 1632 induction supernatants and luciferase reaction systems, and wild-type strains were used as controls. A total of 9 single clones with chemiluminescence values 1.2 times or more than the control group were obtained by preliminary screening (see Figure 3 ), among which the chemiluminescence values of LE30002-H10 and LF23001-F7 were 1.2-1.5 times that of the wild-type luciferase, the chemiluminescence values of LE30001-C12, LF23001-D11, LF23002-C12 and LF23002-D11 were 1.5-2.0 times that of the wild-type luciferase, and the chemiluminescence values of LF23001-C11, LF23001-C12 and LF23002-G11 were more than 2.0 times that of the wild-type luciferase.

[0053] The above 9 single clones were sequenced, and the results showed that the 9 single clones were 4 mutants, among which LE30001-C12 was a mutant named Luc-1, LE30002-H10 was a mutant named Luc-2, LF23001-C11 and LF23001-C12 were the same mutant named Luc-3, and the remaining five mutant strains LF23001-D11, LF23001-F7, LF23002-C12, LF23002-D11 and LF23002-G11 were the same mutant named Luc-4.

[0054] The 4 mutants were re-shaken, and the intensity of the luciferase reaction system catalyzed by the induction supernatant was detected. The results showed that the enzyme activity of the Luc-3 mutant was significantly enhanced, about 2.5 times that of the wild-type luciferase; the enzyme activity of the other three mutants was also enhanced, among which the Luc-1 mutant was about 1.3 times that of the wild-type luciferase, the Luc-2 mutant was about 1.4 times that of the wild-type luciferase, and the Luc-4 mutant was about 1.6 times that of the wild-type luciferase (see Figure 4 ). The sequencing results showed that the 1142th base of the Luc-3 mutant was mutated from cytosine (C) to thymine (T) (see Figure 5), resulting in the mutation of the 381st amino acid from threonine (T) to isoleucine (I), and the corresponding gRNA is gRNA21 shown by the sequence of SEQ ID NO. 23.

[0055] The nucleic acid sequence of the Luc-3 mutant is shown in SEQ ID NO. 2, and the amino acid sequence is shown in SEQ ID NO. 1.

[0056] Example 2: Evaluation of the activity of the luciferase mutant

[0057] To verify the stability of the Luc-3 monoclonal secreted luciferase, three biological repeats were performed, including culture of bacteria, induction, and detection of enzyme activity.

[0058] 10 μL of Luc-3 and wild-type luciferase yeast liquid were added to 1 mL of MGY culture medium, respectively, and cultured at 30°C and 800 rpm for 24 h. The culture medium was removed by centrifugation, and MMY culture medium was added for induction for 24 h. Every 24 h, 0.5% methanol was added, a total of 2 times, and the induction expression was performed for 72 h. The supernatant was collected by centrifugation, and at this time, the supernatant still contained secreted luciferase.

[0059] 10 μL of induced supernatant and 90 μL of luciferase reaction system were added to a 96-well plate, and then the plate was placed in a Glomax instrument with an integration time of 4 s. The chemiluminescence intensity was detected.

[0060] The statistical results of three biological repeats showed that the enzyme activity of the Luc-3 mutant was significantly enhanced, about 3 times that of the control group (see Figure 6 ), which is consistent with the screening results. The specific RLU values of the three biological repeats were 12093, 11760, and 11050 for the wild-type luciferase, and 34000, 34333, and 35844 for the Luc-3 mutant, respectively. The results showed that the Luc-3 mutant had high stability, good repeatability, and stable enzyme activity.

[0061] Example 3: Application of luciferase mutant in detection

[0062] To explore the possibility of application of the luciferase mutant (Luc-3) in detection, we purchased a domestic luciferase detection kit for comparison. The purchased kit was used as the control group, and Luc-3 was used to replace the luciferase in the kit as the experimental group. The results showed that the fluorescence value of Luc-3 was about 1.5 times that of the control group (see Figure 7 ), indicating that Luc-3 has the potential to be developed into a kit. The specific operation is as follows:

[0063] Transfer 10 μL of Luc-3 glycerol bacteria to 1 mL of MGY medium and incubate at 30℃ and 800 rpm for 24 h using a shaker. Centrifuge at 2000 μg for 5 min to remove the supernatant, resuspend in 1 mL of MMY medium, and induce incubation at 28℃ and 800 rpm for 72 h, adding 5 μL of methanol every 24 h. After induction, centrifuge at 2000 μg for 5 min and collect the supernatant, storing it at 4℃ for later use. Take 90 μL of commercial reagent without the original luciferase, add 10 μL of the induction supernatant containing 0.48 μg Luc-3, and add 0.52 μg of the original luciferase to 90 μL of commercial reagent. Add 10 μL of ultrapure water, and add 10 μL of 1 μM ATP to each well of a black 96-well plate. Mix well and immediately use a TECAN multi-mode microplate reader to detect the chemiluminescence value and record the data.

Claims

1. A luciferase mutant, characterized in that, The amino acid sequence of the luciferase mutant is shown in SEQ ID NO.

1.

2. A polynucleotide encoding the luciferase mutant of claim 1.

3. The polynucleotide according to claim 2, characterized in that, The sequence of the polynucleotide is shown in SEQ ID NO.

2.

4. An expression vector containing the polynucleotide of claim 2 or 3.

5. A host cell containing the expression vector of claim 4.

6. The use of the luciferase mutant according to claim 1 in the preparation of a luciferase detection kit.

7. The use of the luciferase mutant of claim 1 as a reporter protein.

8. The use of the polynucleotide of claim 2 or 3 as a luciferase reporter gene.

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