Composition, method and application of bioluminescence-based protein ligase activity determination

Abstract

The present invention relates to a novel method for determining the activity of a protein ligase based on bioluminescence, which is generally applicable to various protein ligases, can be used for the characterization, activity tracking and inhibitor screening of known protein ligases, and can also be used for screening various novel protein ligases from plants and other sources. The present invention is characterized in that the recognition sequence of the protein ligase is fused to the C-terminal of the inactive NanoLuc luciferase large fragment (LgBiT) by recombinant expression or other methods, and the nucleophilic attack sequence recognized by the protein ligase is introduced at the N-terminal of the low-affinity SmBiT complementary tag by chemical synthesis or other methods. The activity determination method is based on bioluminescence detection, and has the advantages of high sensitivity, simple operation, anti-protease interference, and suitability for high-throughput screening, and plays an important role in studying known protein ligases or screening unknown protein ligases.

Description

Technical Field

[0001] The present invention relates to biological detection, enzymology and genetic engineering technology, and in particular to a novel method for determining the activity of various protein ligases based on bioluminescence, including key designs, components, steps and various applications of the method. Background Art

[0002] Protein ligases (or polypeptide ligases) are a special class of proteases that can connect two peptide chains together through peptide bonds or form a cyclic peptide through a peptide bond through a transpeptidation mechanism. Currently, the most studied protein ligases include sortases from Gram-positive bacteria and asparaginyl endopeptidase (AEP)-type protein ligases from some plants, such as Butelase-1 from Clitoria ternatea and OaAEP1 from Oldenlandia affinis. Protein ligases are widely used in the modification of proteins and peptides and the synthesis of cyclic peptides, and have important application development value. Therefore, it is necessary to characterize the activity of known protein ligases and continuously screen and identify various new protein ligases in order to obtain various protein ligases with better performance and more practical applications. Current methods for determining protein ligase activity are usually based on fluorescence detection (fluorescent probe-labeled substrate), high-performance liquid chromatography detection or mass spectrometry detection. These methods all have certain shortcomings, such as low sensitivity, long time consumption, susceptibility to protease interference, the need for expensive instruments and equipment, and difficulty in high-throughput screening. Summary of the invention

[0003] The objects of the present invention are: 1) to provide various ligated LgBiT and ligated SmBiT for protein ligase activity assay, and their design features and preparation methods. 2) to provide a method for carrying out bioluminescence-based assay of various protein ligase activities using ligated LgBiT and ligated SmBiT. 3) to provide multiple uses of the activity assay method, including screening various unknown protein ligases from plants or other sources, and characterizing various known protein ligases.

[0004] The present invention provides a composition, which comprises a linker type LgBiT and a linker type SmBiT. The linker type LgBiT is a protein obtained by fusing a recognition sequence of a protein ligase to be tested to the C-terminus of LgBiT through a linker sequence; the linker type SmBiT is a polypeptide obtained by introducing a nucleophilic attack sequence recognized by the protein ligase to be tested to the N-terminus of SmBiT.

[0005] The background luciferase activity of the linked LgBiT is low, but high luciferase activity can be restored after complementation with HiBiT. Its background luciferase activity and the luciferase activity after HiBiT complementation are close to the corresponding values ​​of the original LgBiT. Generally speaking, at least the background luciferase activity of the linked LgBiT is less than 1% of the high luciferase activity that can be restored after complementation with HiBiT. If the background luciferase activity of the linked LgBiT is less than one thousandth or one ten-thousandth of the high luciferase activity that can be restored after complementation with HiBiT, the effect is better.

[0006] When the protein ligase to be tested is Sortase A, the connection type LgBiT is LgBiT-long-LPETG, and its amino acid sequence is shown in Seq ID NO.6; the connection type SmBiT is 4G-SmBiT, and its amino acid sequence is shown in Seq ID NO.15.

[0007] When the protein ligase to be tested is an asparagine protease-type protein ligase, the ligation-type LgBiT is LgBiT-NHV, whose amino acid sequence is shown in Seq ID NO.8; the ligation-type SmBiT is GI-SmBiT, whose amino acid sequence is shown in Seq ID NO.16.

[0008] When the protein ligase to be tested is a proline protease-type protein ligase, the ligation type LgBiT is LgBiT-PIQ, and its amino acid sequence is shown in Seq ID NO.10; the ligation type SmBiT is FG-SmBiT, and its amino acid sequence is shown in Seq ID NO.17.

[0009] A method for measuring the activity of various protein ligases based on bioluminescence, using the composition.

[0010] The method comprises the steps of mixing the composition with the protein ligase to be tested to carry out a ligation reaction at a relatively high concentration (μmol / l level), then appropriately diluting the reaction mixture to reduce the background activity caused by the bimolecular complementarity of the ligated LgBiT and the ligated SmBiT, and finally carrying out a bioluminescence assay on the diluted reaction solution.

[0011] The application of the composition or the method in any of the following should also be within the protection scope of the present invention:

[0012] 1) Application in the preparation of products for determining protein ligase activity;

[0013] 2) Application in determining protein ligase activity;

[0014] 3) Application in screening protein ligases from plants, animals or microorganisms;

[0015] 4) Application in identifying protein ligases from plants, animals or microorganisms;

[0016] The protein ligase is various types of protein ligases such as AEP type protein ligase, PEP type protein ligase, Sortase, etc.

[0017] The invention provides application of protein ligase activity determination in characterization of protein ligase, activity tracking of protein ligase and screening of inhibitors of protein ligase.

[0018] The present invention provides a new method for determining the activity of a protein ligase based on bioluminescence. The method is generally applicable to various protein ligases, can be used for the characterization, activity tracking and inhibitor screening of known protein ligases, and can also be used for screening various new protein ligases from plants and other sources. The present invention is characterized in that the recognition sequence of the protein ligase is fused to the C-terminus of the inactive NanoLuc luciferase large fragment (LgBiT) by recombinant expression or other methods, and the nucleophilic attack sequence recognized by the protein ligase is introduced to the N-terminus of the low-affinity SmBiT complementary tag by chemical synthesis or other methods. The activity determination method is based on bioluminescence detection, and has the advantages of high sensitivity, simple operation, resistance to protease interference, and suitability for high-throughput screening. It plays an important role in studying known protein ligases or screening unknown protein ligases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 :Principle and operation flow of the new method for determining protein ligase activity based on bioluminescence. (A) General form of ligated LgBiT and ligated SmBiT for determining protein ligase activity. The recognition sequence of the protein ligase is fused to the C-terminus of LgBiT through a suitable ligating peptide, and the nucleophilic attack sequence recognized by the protein ligase is introduced to the N-terminus of SmBiT. (B) General operation steps of the new method for determining protein ligase activity based on bioluminescence. High concentrations of ligated LgBiT and ligated SmBiT are ligated under the catalysis of protein ligase. A small amount of the reaction mixture (such as 10 μl) is taken out at different reaction times for appropriate dilution (usually 100 to 100,000 times), and then a small amount of the diluted sample (such as 10 μl) is taken and added to the NanoLuc substrate and the bioluminescence is measured on an ELISA instrument.

[0020] Figure 2:A new method for determining protein ligase activity based on bioluminescence was validated using bacterial Sortase A. (A) Schematic diagram of ligated LgBiT and SmBiT and their expected ligation products used for Sortase A ligase activity determination. The amino acids are represented by single letters. (B) Luciferase activity and purity of recombinantly expressed ligated LgBiT and expected ligation products were identified by bioluminescence and SDS-PAGE. (C) Changes in bioluminescence with reaction time during the ligation of LgBiT-long-LPETG and 4G-SmBiT catalyzed by recombinant circular Sortase A. Samples (10 μl) were taken at different reaction times and diluted 10,000 times in two steps, and then 10 μl of the diluted samples were taken to determine bioluminescence. (D, E) Detection of the ligation of LgBiT-long-LPETG and 4G-SmBiT catalyzed by Sortase A by bioluminescence and SDS-PAGE. When measuring bioluminescence, samples (10 μl) were taken at different reaction times and diluted 100,000 times in three steps, and then 10 μl of the diluted sample was taken to measure bioluminescence. When SDS-PAGE was detected, samples (10 μl) were taken at different reaction times, mixed with 40 μl SDS-PAGE buffer, boiled, and 10 μl was loaded, electrophoresed, and stained with Coomassie Brilliant Blue. The band of LgBiT-long-LPETG is indicated by an asterisk, and the band of the ligation product is indicated by a pound sign. The band of the ligation product overlaps with the band of the recombinant circular Sortase A.

[0021] Figure 3:Verification of a new method for determining protein ligase activity based on bioluminescence using plant Butelase-1. (A) Schematic diagram of the ligated LgBiT and SmBiT used for Butelase-1 ligase activity determination and the expected ligation products of the two. The amino acids are represented by single letters. (B) The luciferase activity and purity of the recombinantly expressed ligated LgBiT and the expected ligation products were identified by bioluminescence and SDS-PAGE. (C) Changes in bioluminescence during the ligation of LgBiT-NHV and GI-SmBiT catalyzed by the butterfly pea crude extract. Samples (10 μl) were taken at different reaction times and diluted 3000 times in two steps, and then 10 μl of the diluted samples were taken to measure bioluminescence. (D) Partially purified Butelase-1 catalyzed the ligation of LgBiT-NHV and GI-SmBiT. Samples (10 μl) were taken at different reaction times and diluted 10000 times in two steps, and then 10 μl of the diluted samples were taken to measure bioluminescence, and then the concentration of the ligation product in the reaction mixture was calculated based on the specific activity of the ligation product. (E) Bioluminescence and SDS-PAGE detection of the ligation of LgBiT-NHV and GI-SmBiT catalyzed by partially purified Butelase-1. For bioluminescence, samples (10 μl) were taken at different reaction times and diluted 100,000 times in three steps. Then, 10 μl of the diluted sample was taken to measure bioluminescence, and the concentration of the ligation product in the reaction mixture was calculated based on the specific activity of the ligation product. For SDS-PAGE detection, samples (10 μl) were taken at different reaction times, mixed with 20 μl of SDS-PAGE buffer, boiled, and 10 μl was loaded for electrophoresis and stained with Coomassie Brilliant Blue. The ligation product band is indicated by an asterisk.

[0022] Figure 4 :Screening of plants that may express AEP-type protein ligase using LgBiT-NHV and GI-SmBiT. Plant tissues were ground in PBS and centrifuged to obtain the supernatant, which was the crude extract. The crude extract was mixed with LgBiT-NHV and GI-SmBiT in the indicated proportions, incubated at 25°C, and samples (10 μl) were taken at different reaction times and diluted 3000 times in two steps. Then 10 μl of the diluted sample was taken to measure bioluminescence. The photo shows the plant parts used to prepare the crude ligase extract.

[0023] Figure 5: Screening of plants that may express PEP-type protein ligases using LgBiT-PIQ and FG-SmBiT. (A) Amino acid sequences of some cyclic peptide precursors with a Pro residue at the C-terminus of the mature peptide. The mature peptide part of the cyclic peptide is shown in red. (B) Design of ligation-type LgBiT and SmBiT for screening PEP-type protein ligases. The amino acids are represented by single letters. (CF) Determination of the activity of PEP-type protein ligases in crude plant extracts. Plant tissues were ground in PBS and centrifuged to obtain the supernatant, which was the crude extract. The crude extract was mixed with LgBiT-PIQ and FG-SmBiT in the indicated proportions, kept at 25°C, and samples (10 μl) were taken at different reaction times and diluted 2000 times in two steps. Then 10 μl of the diluted sample was taken to measure bioluminescence. The photos show the plant parts used to prepare the crude ligase extract. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0025] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified. If not otherwise specified, the methods listed in the experimental manuals such as "Molecular Cloning Experiment Guide" (3rd edition) (Cold Spring Harbor Laboratory Press), "Cell Experiment Guide" (Science Press, Beijing, China, 2001) and the references cited herein can be used for implementation.

[0026] The pET expression vector in the following embodiments is disclosed in the document Wang JH, Shao XX, Hu MJ, Wei D, Nie WH, Liu YL, Xu ZG, Guo ZY. (2017) Rapid preparation of bioluminescent tracers for relaxin family peptides using sortase-catalyzed ligation. Amino Acids, 49: 2455-2459; the Escherichia coli expression strain BL21 (DE3) is disclosed in the document Wang JH, Shao XX, Hu MJ, Wei D, Nie WH, Liu YL, Xu ZG, Guo ZY. (2017) Rapid preparation of bioluminescent tracers for relaxin family peptides using sortase-catalyzed ligation. Amino Acids, 49: 2455-2459; the public can obtain it from Tongji University for verification of the present invention.

[0027] The 80 plants in the following embodiments were all collected by the inventors themselves, among which the butterfly pea was collected in Hainan, China in March 2021; the other plant samples were collected in Shanghai, China between July and August 2021.

[0028] Bioluminescence refers to a type of chemiluminescent reaction catalyzed by various luciferases. It has the advantages of low detection background, wide linear range, and strong anti-interference ability, and is widely used in various quantitative detections. Luciferases commonly used in bioluminescence assays include firefly luciferase, sea renilla luciferase, and NanoLuc luciferase developed in recent years. Among them, NanoLuc luciferase has the advantages of small weight (171 amino acids), stable structure, and high luminescence efficiency. Based on NanoLuc, Promega has developed NanoLuc Binary Technology (NanoBiT) in recent years, which contains an inactive NanoLuc large fragment (referred to as LgBiT, consisting of 159 amino acid residues) and two complementary tags, namely the low-affinity SmBiT tag and the high-affinity HiBiT tag, both of which have only 11 amino acid residues. The low-affinity LgBiT and SmBiT systems are often used to study the dynamic interactions between two protein molecules, while the high-affinity LgBiT and HiBiT systems can be used to quantify HiBiT-tag fused proteins or other molecules.

[0029] Due to the low affinity (K d The value is 190 μmol / l). Only at high concentrations can SmBiT effectively achieve bimolecular complementarity with LgBiT to restore the luciferase activity. It is speculated that if SmBiT can be covalently linked to the C-terminus of LgBiT, the linked SmBiT can not only effectively restore the activity of LgBiT, but also will not be affected by free SmBiT. Based on this, the present invention uses LgBiT and SmBiT to develop a new method for determining the activity of various protein ligases, that is, a recognition sequence of a certain protein ligase or a certain type of protein ligase is fused to the C-terminus of LgBiT through a linker sequence, and a nucleophilic attack sequence recognized by this or this type of protein ligase is introduced to the N-terminus of SmBiT ( Figure 1 A). If the ligated LgBiT and ligated SmBiT can be linked together by the expected protein ligase, the ligation product can restore the luciferase activity, and after adding the NanoLuc substrate, it can be quantitatively determined by bioluminescence, and the detection limit can reach the fmol level or even lower.

[0030] The present invention provides a connected LgBiT and a connected SmBiT, and design points and preparation methods thereof.

[0031] Design points and preparation method of ligated LgBiT: The recognition sequence of a known protein ligase or a certain type of protein ligase or the inferred recognition sequence of a certain protein ligase or a certain type of protein ligase is fused to the C-terminus of the original LgBiT protein through a suitable connecting peptide. Figure 1 A). By changing the length or amino acid composition of the connecting peptide, the background luciferase activity of the connected LgBiT is ensured to be low, and the high luciferase activity can be restored after HiBiT complementation. For example, in the present invention, by introducing the known Sortase A recognition sequence and optimizing its connecting peptide, LgBiT-long-LPETG with low background luciferase activity for Sortase A ligase activity determination is obtained; by introducing the known Butelase-1 recognition sequence, LgBiT-NHV for determining Butelase-1 protein ligase activity and screening asparagine protease (AEP) type protein ligase is obtained; by introducing the presumed proline amino acid protease (PEP) type protein ligase recognition sequence, LgBiT-PIQ for screening PEP type protein ligase is obtained. Other researchers can easily design a connected LgBiT for various protein ligase activity determinations based on the design points provided in this patent.

[0032] The present invention establishes a method for preparing various linked LgBiTs by recombinant expression in Escherichia coli, and optimizes the induction conditions, including the induction temperature and the inducer concentration. In order to facilitate the purification of various linked LgBiTs, a 6xHis tag is fused to its N-terminus, so that the recombinantly expressed LgBiT can be purified by solid phase metal chelate chromatography (Ni 2+ Column) purification. Then, after a step of anion exchange chromatography, various linked LgBiTs with a single band can be obtained. In order to determine whether the designed linked LgBiT is usable, it is necessary to measure its background luciferase activity and the luciferase activity after HiBiT complementation.

[0033] Design points and preparation methods of ligated SmBiT: A nucleophilic attack sequence recognized by a certain protein ligase or a certain type of protein ligase or a presumed nucleophilic attack sequence of a certain protein ligase or a certain type of protein ligase is introduced into the N-terminus of the original SmBiT. A suitable linker ( Figure 1 A). For example, in the present invention, four consecutive Gly residues are introduced as the affinity attack sequence recognized by Sortase A, and 4G-SmBiT for measuring the activity of Sortase A is obtained; by introducing Gly-Ile as the affinity attack sequence recognized by Butelase-1, and adding two Gly as a linker, GI-SmBiT for measuring the activity of Butelase-1 and screening AEP type protein ligase is obtained; by introducing Phe as the affinity attack sequence recognized by PEP type protein ligase, and adding three Gly as a linker, FG-SmBiT for screening PEP type protein ligase is obtained. Other researchers can easily design a connection type SmBiT for measuring the activity of various protein ligases based on the design points provided by this patent.

[0034] Various linker-type SmBiTs can be chemically synthesized by a mature solid-phase peptide synthesis process, and then purified by high performance liquid chromatography (HPLC) using a C18 reverse phase column and confirmed by molecular weight determination by mass spectrometry.

[0035] The present invention provides a method for measuring the activities of various protein ligases based on bioluminescence by utilizing a ligated LgBiT and a ligated SmBiT.

[0036] In order to use ligated LgBiT and ligated SmBiT to carry out the activity assay of various protein ligases, a simple and reasonable operation process was developed, namely, the three steps of ligation reaction, dilution of ligation reaction mixture, and bioluminescence assay ( Figure 1B). Protein ligases usually require a high substrate concentration to effectively catalyze the ligation reaction, so a high concentration (μmol / l level) of ligation-type LgBiT and ligation-type SmBiT is used in the ligation reaction. However, at high concentrations, LgBiT and SmBiT can undergo bimolecular complementation, resulting in strong background luminescence, making it impossible to measure a small amount of ligation products (Table 1). A key strategy for the success of this method is to appropriately dilute the ligation reaction mixture ( Figure 1 B), during the dilution process, the background luminescence generated by the bimolecular complementation of LgBiT and SmBiT will decrease according to the square of the dilution multiple, while the signal luminescence generated by the ligation product will decrease linearly according to the dilution multiple (Table 1), so under appropriate dilution conditions, the luminescence generated by the ligation product will be significantly greater than the background luminescence. Under appropriate dilution conditions, even if only one thousandth of the ligation-type LgBiT is catalyzed by the protein ligase to connect with the ligation-type SmBiT, the ligation product can be sensitively detected (Table 1). According to the three designed experimental steps, the activity of various known or unknown protein ligases can be determined using the corresponding ligation-type LgBiT and ligation-type SmBiT.

[0037] Table 1 shows the background luminescence generated by the bimolecular complementation of LgBiT and SmBiT and the signal luminescence generated by the ligation product under different dilution conditions obtained by theoretical calculation. d The K values ​​of the original LgBiT and the original SmBiT are combined d The value is the same, i.e., 190 μmol / l. Assume that 1% or 0.1% of the ligated LgBiT and SmBiT in the ligation reaction are ligated together by protein ligase. Assume that the luciferase specific activity of the ligation product and the SmBiT complementary LgBiT is 5×10 5 RLU / fmol (measured on iD3 microplate reader using 384-well plate). After the ligation reaction starts, samples are taken at different reaction times and appropriately diluted, and then the diluted samples are taken into 384-well plates (10μl / well). Bioluminescence is measured on a microplate reader immediately after adding diluted NanoLuc luciferase substrate (10μl / well). Red indicates that the signal luminescence is significantly higher than the background luminescence under this dilution condition.

[0038] Table 1: Background luminescence generated by LgBiT and SmBiT bimolecular complementation and signal luminescence generated by the ligation product under different dilution conditions obtained by theoretical calculation

[0039]

[0040] The invention provides an application of a method for measuring protein ligase activity based on bioluminescence.

[0041] The method based on bioluminescence to determine the activity of protein ligase has the advantages of high sensitivity, simple operation, resistance to protease interference, and suitability for high-throughput screening. It has a wide range of application prospects in protein ligase research, including screening and identification of various unknown protein ligases from plants or other sources, as well as related research on various known protein ligases. For example, by screening crude extracts of 80 common plants, it was determined that 5 plants, including Zephyranthes candida, Hibiscus syriacus, Angelonia angustifolia, Hosta plantaginea, and Jasminum nudiflorum, may express AEP-type protein ligases; 4 plants, including Angelonia angustifolia, Aucuba japonica var.variegate, Eupatorium fortunei, and Lindernia antipoda, may express PEP-type protein ligases. The activity assay method can be used to quickly and cheaply screen a large number of plants, animals and microorganisms to identify various types of protein ligases. In addition, the activity assay method can also be used for characterization of known protein ligases, activity tracking, inhibitor screening and other research.

[0042] Example 1: Verification of a new method for determining protein ligase activity based on bioluminescence using bacterial Sortase A

[0043] 1. Design of ligated LgBiT and SmBiT for determination of Sortase A ligase activity:

[0044] It is known that the recognition sequence of Sortase A consists of 5 amino acid residues, usually LPETG, so this sequence was introduced into the C-terminus of LgBiT, and an extra Gly was added after it for protection ( Figure 2 A). At the same time, we tried to introduce different lengths of connecting peptides between LgBiT and the recognition sequence to ensure that the background luciferase activity of the obtained connected LgBiT was low, but the luciferase activity could be restored after complementation with HiBiT. When the connecting peptide was 3 Gly residues, the obtained connected LgBiT was named LgBiT-LPETG, and its amino acid sequence was shown in Seq ID NO.4; when the connecting peptide was 18 amino acid residues, it was named LgBiT-long-LPETG, and its amino acid sequence was shown in Seq ID NO.6.

[0045] The nucleophilic attack sequence recognized by Sortase A is usually a series of consecutive Gly residues, so four consecutive Gly residues were introduced at the N-terminus of SmBiT, named 4G-SmBiT ( Figure 2 A), whose sequence is shown in Seq ID NO.16.

[0046] In addition, the expected ligation product of LgBiT-long-LPETG and 4G-SmBiT was designed and named LgBiT-long-LPET-4G-SmBiT, whose amino acid sequence is shown in Seq ID NO.12, to verify whether the expected ligation product has luciferase activity ( Figure 2 A).

[0047] 2. Chemical synthesis and purification of 4G-SmBiT:

[0048] 4G-SmBiT was chemically synthesized by conventional solid phase peptide synthesis methods, and the synthesized crude peptide was purified by high performance liquid chromatography (HPLC) using a C18 reverse phase column to obtain a pure sample, and the mass spectrum showed that its molecular weight was correct. After purification, the freeze-dried 4G-SmBiT sample was dissolved in 1.0 mmol / l dilute hydrochloric acid (pH 3.0), and the concentration was determined by 280 nm ultraviolet absorption (extinction coefficient was 1490 l / mol / cm), and then divided into portions (20-50 μl / tube) and frozen at -80°C for subsequent experiments.

[0049] 2. Recombinant expression and purification of various LgBiT proteins and their fusion proteins:

[0050] 1. Preparation of recombinant vector

[0051] 1.1 The coding sequence of chemically synthesized LgBiT (as shown in Seq ID NO.1) was used to replace the sequence between the NdeI and EcoRI sites in the pET expression vector, and other sequences were kept unchanged. The resulting recombinant vector was pET / LgBiT, which encodes an LgBiT protein with a 6xHis tag at the N-terminus (as shown in Seq ID NO.2).

[0052] 1.2 Replace the coding sequence of LgBiT in step 1.1 with LgBiT-LPETG (as shown in Seq ID NO.3), and keep other steps unchanged to obtain the recombinant vector pET / LgBiT-LPETG, which encodes LgBiT-LPETG protein (as shown in Seq ID NO.4).

[0053] 1.3 Replace the coding sequence of LgBiT in step 1.1 with LgBiT-long-LPETG (as shown in Seq ID NO.5), and keep other steps unchanged to obtain the recombinant vector pET / LgBiT-long-LPETG, which encodes LgBiT-long-LPETG protein (as shown in Seq ID NO.6).

[0054] 1.4 Replace the coding sequence of LgBiT in step 1.1 with LgBiT-long-LPET-4G-SmBiT (as shown in Seq ID NO.13), and keep other steps unchanged to obtain the recombinant vector pET / LgBiT-long-LPET-4G-SmBiT, which encodes LgBiT-long-LPET-4G-SmBiT protein (as shown in Seq ID NO.14).

[0055] The coding sequences of LgBiT-LPETG, LgBiT-long-LPETG and LgBiT-long-LPET-4G-SmBiT can be obtained by PCR amplification or chemical synthesis. The coding sequences of various LgBiTs are verified by DNA sequencing.

[0056] 2. Protein Preparation and Purification

[0057] 2.1 Preparation of LgBiT protein

[0058] The recombinant expression vector pET / LgBiT was transformed into the Escherichia coli expression strain BL21(DE3) to obtain the transformed strain BL21(DE3)-pET / LgBiT; the transformed strain BL21(DE3)-pET / LgBiT was cultured in LB liquid culture medium (containing 100mg / l ampicillin) in a 37°C shaker until the optical density OD600 reached 1-2. Then the culture solution was cooled to 16°C, the inducer IPTG was added to 0.5mmol / l, and the culture was continued to be slowly shaken in a 16°C shaker overnight. Then centrifugation (5000g, 10min) was performed to collect the bacteria, and the bacteria were resuspended with an appropriate amount of lysis buffer (20mmol / l phosphate, pH7.4, 0.5mol / l NaCl), and the bacteria were lysed by ultrasound. Then centrifugation (12000g, 20min), the supernatant was taken, and Ni 2+Column purification. The LgBiT protein was eluted with 250mmol / l imidazole (dissolved in lysis buffer), and the eluted fractions were collected and dialyzed against 10mmol / l Tris-Cl (pH7.5) solution overnight. After dialysis, centrifugation (10000g, 10min) was performed, and the supernatant was purified by EDAE ion exchange chromatography and eluted with a NaCl linear gradient. The eluent A was 10mmol / l Tris-Cl (pH7.5), and the eluent B was 500mmol / lNaCl added to the eluent A. The LgBiT protein elution peak was collected and identified as a single band by SDS-PAGE, and the size was consistent with the expected ( Figure 2 B). The purified LgBiT was quantified by 280 nm UV absorption (the extinction coefficient of LgBiT is 20000 l / mol / cm), and then aliquoted (20-50 μl / tube) to obtain LgBiT protein, which was frozen at -80°C for subsequent experiments.

[0059] 2.2 Preparation of LgBiT-LPETG protein

[0060] The recombinant expression vector pET / LgBiT in step 2.1 was replaced with pET / LgBiT-LPETG, and the other steps remained unchanged to obtain the transformed strain BL21(DE3)-pET / LgBiT-LPETG, and further obtain the LgBiT-LPETG protein.

[0061] 2.3 Preparation of LgBiT-long-LPETG protein

[0062] The recombinant expression vector pET / LgBiT in step 2.1 was replaced with pET / LgBiT-long-LPETG, and the other steps remained unchanged to obtain the transformed strain BL21(DE3)-pET / LgBiT-long-LPETG, and further obtain the LgBiT-long-LPETG protein.

[0063] 2.4 Preparation of LgBiT-long-LPET-4G-SmBiT protein

[0064] The recombinant expression vector pET / LgBiT in step 2.1 was replaced with LgBiT-long-LPET-4G-SmBiT, and the other steps remained unchanged to obtain the transformed strain BL21(DE3)-pET / LgBiT-long-LPET-4G-SmBiT, and further obtain the LgBiT-long-LPET-4G-SmBiT protein.

[0065] 3. Luciferase activity assay of various proteins:

[0066] 3.1 Determination of background luciferase activity of proteins

[0067] The LgBiT, LgBiT-long-LPETG and LgBiT-long-LPET-4G-SmBiT proteins in step 2 were gradiently diluted, and each protein was diluted to 10 nmol / l, 1.0 nmol / l and 0.1 nmol / l, respectively. The diluent was phosphate buffered saline (PBS) added with 0.1% bovine serum albumin (BSA) and 0.01% TWEEN-20.

[0068] The above diluted samples were taken into 384-well plates (10 μl / well), and the NanoLuc substrate stock solution purchased from Promega was diluted 100 times with the above diluent, and then added to the 384-well plate (10 μl / well). After mixing, the bioluminescence was immediately measured on a microplate reader. The background luciferase activity of LgBiT-LPETG was very high (reaching about 30% of the activity after HiBiT complementation), indicating that it was not suitable for use as a substrate for Sortase A. The background luciferase activity of LgBiT-long-LPETG was very low, similar to the background luciferase activity of the original LgBiT ( Figure 2 B), indicating that LgBiT-long-LPETG can be used as a substrate for Sortase A. The expected ligation product LgBiT-long-LPET-4G-SmBiT itself has high luciferase activity ( Figure 2 A), the specific activity was determined to be 4.1×10 5 RLU / fmol, which is only slightly lower than the activity of HiBiT-complemented LgBiT-long-LPETG. Therefore, once the recombinantly expressed LgBiT-long-LPETG and chemically synthesized 4G-SmBiT are ligated together by Sortase A, a ligation product with high luciferase activity will be obtained.

[0069] 3.2 Determination of protein luciferase activity after HiBiT complementation

[0070] The LgBiT and LgBiT-long-LPETG proteins in step 2 were diluted to 20 nmol / l, 2.0 nmol / l and 0.2 nmol / l respectively with the above diluent, and then these diluted samples were mixed with an equal volume of 80 nmol / l HiBiT (prepared with the above diluent), placed at room temperature for 10 minutes, and then added to a 384-well plate (10 μl / well), and diluted NanoLuc substrate (10 μl / well) was added. After mixing, the bioluminescence was immediately measured on an ELISA reader. The results are as follows Figure 2 As shown in B, after complementation with HiBiT, the luciferase activity of LgBiT-long-LPETG was restored ( Figure 2 B), the specific activity was determined to be 5.5×105 RLU / fmol, which is almost the same as the luciferase activity after complementation of the original LgBiT with HiBiT.

[0071] 4. Recombinant expression and purification of circular Sortase A:

[0072] Sortase A is derived from Staphylococcus aureus, and its active part consisting of amino acids 60-206 is recombinantly expressed. According to the method previously reported by our laboratory (see reference Hu MJ, Shao XX, Li HZ, Nie WH, Wang JH, Liu YL, Xu ZG, Guo ZY. (2018) Development of a novel ligand binding assay for relaxin family peptide receptor 3 and 4 using NanoLuc complementation. Amino Acids, 50: 1111-1119), the amino acid residues 60-206 of Sortase A were fused with a split intein, which can catalyze the formation of a peptide bond between the N-terminus and the C-terminus of linear Sortase A, thereby obtaining a more stable circular Sortase A. After inducing expression in Escherichia coli, the product was purified by DEAE anion exchange chromatography and SP cation exchange chromatography. The resulting circular Sortase A was identified as a single band by SDS-PAGE and quantified by 280 nm UV absorption (extinction coefficient 13490 l / mol / cm), then aliquoted (20-50 μl / tube) and frozen at -80°C for subsequent experiments to obtain recombinant circular Sortase A.

[0073] 5. Bioluminescence assay of recombinant cyclic Sortase A catalyzing the ligation of LgBiT-long-LPETG and 4G-SmBiT:

[0074] The preheated LgBiT-long-LPETG, 4G-SmBiT and recombinant circular Sortase A were mixed in a ligation buffer (100 mmol / l Tris-Cl, pH 7.5, 10 mmol / l CaCl2, 0.1% BSA) in the mixing ratio shown in Table 2, and the ligation reaction was carried out at 25°C.

[0075] Table 2 Mixing ratio of LgBiT-long-LPETG, 4G-SmBiT and recombinant circular Sortase A

[0076] LgBiT-long-LPETG (μmol / l) 4G-SmBiT (μmol / l) Sortase A (μmol / l) 1 50 200 0 2 50 200 4 3 50 200 8 4 50 200 16 5 0 200 16 6 50 0 16

[0077] At different reaction times (0 min, 5 min, 10 min, 15 min, 20 min, 25 min), 10 μl of the reaction mixture was taken out and added to 990 μl of diluent (PBS with 0.1% BSA and 0.01% TWEEN-20) for 100-fold dilution. Then, 10 μl of the diluted reaction mixture was taken out and added to 990 μl of diluent for 100-fold dilution to obtain a 10,000-fold diluted reaction mixture. The 10,000-fold diluted reaction mixture was added to a 384-well plate (10 μl / well), and then the NanoLuc substrate diluted 100-fold with the diluent (10 μl / well) was added. After mixing, the bioluminescence was immediately measured on an ELISA reader. Figure 2 As shown in C, when LgBiT-long-LPETG, 4G-SmBiT and recombinant circular Sortase A are all present, the measured bioluminescence increases linearly with the reaction time, and the rate of increase in luminescence is proportional to the concentration of Sortase A used. However, when any of the three is missing, no luminescence change is observed ( Figure 2 C). This indicates that recombinant circular Sortase A can catalyze the ligation of LgBiT-long-LPETG and 4G-SmBiT to generate a ligation product with luciferase activity.

[0078] Further, a long-term ligation reaction was performed with a high-concentration substrate, and samples were taken at different reaction times and diluted 100,000 times to measure bioluminescence, and the concentration of the ligation product in the reaction mixture was calculated based on the specific activity of the ligation product. Figure 2 As shown in D, the concentration of the ligation product reached the highest level at 6 h, about 40 μmol / l, that is, about 80% of LgBiT-long-LPETG and 4G-SmBiT were connected together. The ligation reaction mixture was detected by SDS-PAGE. The results are shown in Figure 2 As shown in Figure E, it was found that the brightness of the LgBiT-long-LPETG band (indicated by the asterisk) gradually decreased with the reaction time, while the brightness of a slightly larger band (indicated by the well sign, which is the connecting product band and the circular Sortase A band, the two overlap) gradually increased with the reaction time ( Figure 2 E), indicating that Sortase A can indeed catalyze the ligation of LgBiT-long-LPETG and 4G-SmBiT.

[0079] Example 2: Verification of a new method for determining protein ligase activity based on bioluminescence using plant Butelase-1

[0080] 1. Design of ligated LgBiT and SmBiT for determination of Butelase-1 ligase activity:

[0081] According to the recognition sequence of Butelase-1, which is three amino acids of NHV, a linker-type LgBiT targeting Butelase-1 was designed. The recognition sequence was introduced into the C-terminus of LgBiT, and a linker peptide consisting of three Gly was added ( Figure 3 A), named LgBiT-NHV, its amino acid sequence is shown in Seq ID NO.8 in the sequence table, and its encoding nucleotide sequence is shown in Seq ID NO.7 in the sequence table. According to the nucleophilic attack sequence of Butelase-1, GI, these two amino acids were introduced into the N-terminus of SmBiT, and two Gly were added as linkers, named GI-SmBiT ( Figure 3 A), whose amino acid sequence is shown in Seq ID NO.16 in the sequence table. The expected ligation product of LgBiT-NHV and GI-SmBiT is named LgBiT-NGI-SmBiT ( Figure 3 A), its amino acid sequence is shown in Seq ID NO.14 in the sequence listing, and its encoding nucleotide sequence is shown in Seq ID NO.13 in the sequence listing.

[0082] 2. Protein Preparation and Purification

[0083] The chemical synthesis and separation and purification of GI-SmBiT were carried out in accordance with Example 1. The expression vector construction, recombinant expression and separation and purification of LgBiT-NHV and LgBiT-NGI-SmBiT were carried out in accordance with Example 1. After purification, LgBiT-NHV and LgBiT-NGI-SmBiT were single bands on SDS-PAGE, and the sizes were in line with expectations ( Figure 3 B).

[0084] 3. Determination of luciferase activity of protein:

[0085] The luciferase activity of LgBiT-NHV and LgBiT-NGI-SmBiT was determined by referring to the method in Example 1. Figure 3 As shown in B, the results show that the recombinant LgBiT-NGI-SmBiT itself has a high luciferase activity ( Figure 3 B), the specific activity was determined to be 4.9×10 5 RLU / fmol. However, the luciferase activity of LgBiT-NHV itself is very low, and the luciferase activity can only be restored after complementation with HiBiT ( Figure 3 B), the specific activity was determined to be 5.6×10 5 RLU / fmol. Therefore, LgBiT-NHV and GI-SmBiT are suitable as Butelase-1 ligation reaction substrates.

[0086] 4. Preparation of butterfly pea crude extract and determination of ligase activity:

[0087] Preparation of Crude Extract of Butterfly Pea

[0088] Butelase-1 is derived from the tropical plant C.ternatea. Weigh 1g of C.ternatea leaves and shoots, chop them into pieces, add 4ml of extract solution and grind them thoroughly in a mortar, then centrifuge (10000g, 5min), and take the supernatant, which is the C.ternatea crude extract. Use PBS and reducing reaction solution (20mmol / l phosphate, pH6.5, 1.0mmol / l EDTA, 5.0mmol / l β-mercaptoethanol) as extracts to prepare C.ternatea crude extract for ligase activity determination.

[0089] The preheated LgBiT-NHV, GI-SmBiT and butterfly pea crude extract were mixed in BPS and reducing reaction solution respectively (mixing ratio is shown in Table 3), and the ligation reaction was carried out at 25°C. At different reaction times (0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min), 10 μl of the reaction mixture was taken out and diluted 3000 times, and then the bioluminescence was measured. The results are shown in Figure 3 C. The bioluminescence measured in the reaction groups of LgBiT-NHV, GI-SmBiT and butterfly pea crude extract gradually increased over a period of time, regardless of whether the reaction was in PBS or in a reducing reaction solution ( Figure 3 C). However, no increase in luminescence was observed in the control group lacking GI-SmBiT or butterfly pea crude extract ( Figure 3 C). Therefore, it is speculated that Butelase-1 in the C. cerevisiae extract can catalyze the connection of LgBiT-NHV and GI-SmBiT, thereby generating a connection product with luciferase activity. However, when the proportion of C. cerevisiae extract in the reaction group was high, the bioluminescence gradually decreased in the later stage ( Figure 3 C), the highest conversion rate of LgBiT-NHV was only about 2%, which may be caused by the hydrolysis of the ligation products by some proteases in the crude extract.

[0090] Table 3 Mixing ratio of LgBiT-NHV, GI-SmBiT and butterfly pea crude extract

[0091] LgBiT-NHV (μmol / l) GI-SmBiT (μmol / l) Extract (reaction volume fraction) 1 5 10 1 / 2 2 5 10 1 / 8 3 5 10 1 / 32 4 5 10 0 5 5 0 1 / 2

[0092] 5. Preparation of partially purified Butelase-1 and determination of ligase activity:

[0093] Butelase-1 was purified from the crude extract of butterfly pea according to the method previously reported (Nguyen GK, Wang S, Qiu Y, Hemu X, Lian Y, Tam JP. (2014) Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nat Chem Biol 10: 732-738. Hemu X, Zhang X, Bi X, Liu CF, Tam JP. (2019) Butelase1-Mediated Ligation of Peptides and Proteins. Methods Mol Biol 2012: 83-109), and partially purified Butelase-1 was obtained after cationic chromatography purification, and the content of Butelase-1 was about 20% as determined by SDS-PAGE. The preheated LgBiT-NHV, GI-SmBiT and partially purified Butelase-1 were mixed in PBS containing 0.1% BSA and the ligation reaction was carried out at 25°C. At different reaction times, 10 μl of the reaction mixture was taken out and diluted 10,000 times, and then 10 μl was taken out to measure bioluminescence. Figure 3 As shown in D, the concentration of the reaction product increased linearly within 25 min, especially in the group with low Butelase-1 concentration. In the group with the highest Butelase-1 concentration, the concentration of the ligation product reached about 0.6 μmol / l at 25 min, that is, the conversion rate of LgBiT-NHV was about 12%.

[0094] Further long-term ligation reaction was performed with high-concentration substrate, and bioluminescence assay showed that the concentration of the ligation product reached about 14 μmol / l ( Figure 3 E), that is, the conversion rate of LgBiT-NHV reached about 60%. SDS-PAGE detection found that the brightness of the LgBiT-NHV band gradually decreased during the reaction, while the brightness of a slightly larger band (indicated by the asterisk, which is the ligation product) gradually increased ( Figure 3 E), indicating that Butelase-1 can indeed catalyze the ligation of LgBiT-NHV and GI-SmBiT.

[0095] Example 3 Screening of new AEP-type protein ligases using a method based on bioluminescence to measure protein ligase activity

[0096] Butelase-1 belongs to asparaginyl endopeptidase (AEP) type protein ligase. In order to screen out new AEP type protein ligase for application development, the present invention establishes an efficient method for screening AEP type protein ligase from plant crude extract or other sources.

[0097] In this example, leaves or flowers of 80 common plants were collected by ourselves, 1g of plant tissue was weighed, 2ml of PBS was added for grinding, and then centrifuged to obtain the supernatant, which was the crude extract. LgBiT-NHV and GI-SmBiT were used as substrates, and the crude extracts of these 80 plants were screened according to the ratio in Table 4. The results are shown in Table 5. After the first round of screening, possible positive samples (bioluminescence increased by more than 2 times after 5 minutes or 10 minutes of reaction compared to 0 minutes) were verified. Figure 4 As shown, this example found significant AEP-type protein ligase activity in crude extracts of five plants, including Zephyranthes candida, Hibiscus syriacus, Angelonia angustifolia, Hostaplantaginea, and Jasminum nudiflorum. However, these protein ligases may only be expressed in specific tissues of these plants. For example, high AEP-type protein ligase activity was detected in the flowers and pedicels of Zephyranthes candida, but very low activity in its leaves and corms; high AEP-type protein ligase activity was detected in the flowers of Hibiscus syriacus, but very low activity in its leaves, indicating that protein ligases in some plants are only expressed in specific parts, and protein ligases can only be identified and purified from these tissues.

[0098] Table 4 Mixing ratio of LgBiT-NHV, GI-SmBiT and plant crude extract

[0099] LgBiT-NHV (μmol / l) GI-SmBiT (μmol / l) Extract (volume fraction) 1 5 10 1 / 2 2 5 10 1 / 4 4 5 10 0 5 5 0 1 / 2

[0100] Table 5 Test results of different test parts of different plants

[0101]

[0102]

[0103]

[0104] Example 4 Screening of PEP-type protein ligases using a method based on bioluminescence to measure protein ligase activity

[0105] In addition to AEP-type protein ligases, there should be other types of protein ligases, because the C-terminus of the mature peptide of some cyclic peptides is not an Asn or Asp residue. Figure 5 As shown in A, the C-terminal of some cyclic peptide mature peptides is a Pro residue, and it is speculated that these cyclic peptides may be synthesized by proline amino acid protease (PEP) type protein ligase catalyzed.

[0106] In order to detect whether there are PEP-type protein ligases in plants, such as Figure 5 As shown in B, in this example, a PIQ sequence is introduced into the C-terminus of LgBiT through a connecting peptide consisting of three Gly as a possible recognition site for PEP-type protein ligase, and it is named LgBiT-PIQ, and its amino acid sequence is shown in Seq ID NO.10 in the sequence table, and its coding sequence is shown in Seq ID NO.9 in the sequence table. A hydrophobic Phe and three Gly residues are introduced into the N-terminus of SmBiT as a nucleophilic attack sequence that may be recognized by PEP-type protein ligase, and it is named FG-SmBiT, and its amino acid sequence is shown in SeqID NO.17 in the sequence table.

[0107] LgBiT-PIQ and FG-SmBiT were prepared according to the method in Example 1, and the background luciferase activity of LgBiT-PIQ and the luciferase activity after complementation with HiBiT were measured. The results showed that the background luciferase activity of LgBiT-PIQ was very low, but the luciferase activity could be restored after complementation with HiBiT, and the measured specific activity was 4.3×10 5 RLU / fmol.

[0108] The 80 plant crude extracts in Example 3 were screened using LgBiT-PIQ and FG-SmBiT in different ratios (see Table 6). Figure 5 CF), found that 4 plants have significant PEP-type protein ligase activity, including Angelonia angustifolia, Aucuba japonica var.variegate, Eupatorium fortunei and Lindernia antipoda. Therefore, the method based on bioluminescence to determine the activity of protein ligase can be used to quickly screen and identify various types of protein ligases from plants or other sources.

[0109] Table 6 Mixing ratio of LgBiT-PIQ, FG-SmBiT and plant crude extract

[0110] LgBiT-PIQ (μmol / l) FG-SmBiT (μmol / l) Extract (volume fraction) 1 5 10 1 / 2 2 5 10 1 / 4 4 5 10 0 5 5 0 1 / 2

[0111] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims. Sequence Listing <110> Tongji University <120> Composition, method and application of bioluminescence-based protein ligase activity assay <160> 17 <170> SIPOSequenceListing 1.0 <210> 1 <211> 507 <212> DNA <213> Artificial Sequence <400> 1 atgcatcacc atcaccacca tatggtcttc acactcgaag atttcgttgg ggactgggaa 60 cagacagccg cctacaacct ggaccaagtc cttgaacagg gaggtgtgtc cagtttgctg 120 cagaatctcg ccgtgtccgt aactccgatc caaaggattg tccggagcgg tgaaaatgcc 180 ctgaagatcg acatccatgt catcatcccg tatgaaggtc tgagcgccga ccaaatggcc 240 cagatcgaag aggtgtttaa ggtggtgtac cctgtggatg atcatcactt taaggtgatc 300 ctgccctatg gcacactggt aatcgacggg gttacgccga acatgctgaa ctatttcgga 360 cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg 420 tggaacggca acaaaattat cgacgagcgc ctgatcaccc ccgacggctc catgctgttc 480 cgagtaacca tcaacagtta agaattc 507 <210> 2 <211> 166 <212> PRT <213> Artificial Sequence <400> 2 Met His His His His His His Met Val Phe Thr Leu Glu Asp Phe Val 1 5 10 15 Gly Asp Trp Glu Gln Thr Ala Ala Tyr Asn Leu Asp Gln Val Leu Glu 20 25 30 Gln Gly Gly Val Ser Ser Leu Leu Gln Asn Leu Ala Val Ser Val Thr 35 40 45 Pro Ile Gln Arg Ile Val Arg Ser Gly Glu Asn Ala Leu Lys Ile Asp 50 55 60 Ile His Val Ile Ile Pro Tyr Glu Gly Leu Ser Ala Asp Gln Met Ala 65 70 75 80 Gln Ile Glu Glu Val Phe Lys Val Val Tyr Pro Val Asp Asp His His 85 90 95 Phe Lys Val Ile Leu Pro Tyr Gly Thr Leu Val Ile Asp Gly Val Thr 100 105 110 Pro Asn Met Leu Asn Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val 115 120 125 Phe Asp Gly Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn 130 135 140 Lys Ile Ile Asp Glu Arg Leu Ile Thr Pro Asp Gly Ser Met Leu Phe 145 150 155 160 Arg Val Thr Ile Asn Ser 165 <210> 3 <211> 534 <212> DNA <213> Artificial Sequence <400> 3 atgcatcacc atcaccacca tatggtcttc acactcgaag atttcgttgg ggactgggaa 60 cagacagccg cctacaacct ggaccaagtc cttgaacagg gaggtgtgtc cagtttgctg 120 cagaatctcg ccgtgtccgt aactccgatc caaaggattg tccggagcgg tgaaaatgcc 180 ctgaagatcg acatccatgt catcatcccg tatgaaggtc tgagcgccga ccaaatggcc 240 cagatcgaag aggtgtttaa ggtggtgtac cctgtggatg atcatcactt taaggtgatc 300 ctgccctatg gcacactggt aatcgacggg gttacgccga acatgctgaa ctatttcgga 360 cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg 420 tggaacggca acaaaattat cgacgagcgc ctgatcaccc ccgacggctc catgctgttc 480 cgagtaacca tcaacagtgg cggtggcctg ccggaaaccg gtggctaaga attc 534 <210> 4 <211> 175 <212> PRT <213> Artificial Sequence <400> 4 Met His His His His His His Met Val Phe Thr Leu Glu Asp Phe Val 1 5 10 15 Gly Asp Trp Glu Gln Thr Ala Ala Tyr Asn Leu Asp Gln Val Leu Glu 20 25 30 Gln Gly Gly Val Ser Ser Leu Leu Gln Asn Leu Ala Val Ser Val Thr 35 40 45 Pro Ile Gln Arg Ile Val Arg Ser Gly Glu Asn Ala Leu Lys Ile Asp 50 55 60 Ile His Val Ile Ile Pro Tyr Glu Gly Leu Ser Ala Asp Gln Met Ala 65 70 75 80 Gln Ile Glu Glu Val Phe Lys Val Val Tyr Pro Val Asp Asp His His 85 90 95 Phe Lys Val Ile Leu Pro Tyr Gly Thr Leu Val Ile Asp Gly Val Thr 100 105 110 Pro Asn Met Leu Asn Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val 115 120 125 Phe Asp Gly Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn 130 135 140 Lys Ile Ile Asp Glu Arg Leu Ile Thr Pro Asp Gly Ser Met Leu Phe 145 150 155 160 Arg Val Thr Ile Asn Ser Gly Gly Gly Leu Pro Glu Thr Gly Gly 165 170 175 <210> 5 <211> 581 <212> DNA <213> Artificial Sequence <400> 5 atgcatcacc atcaccacca tatggtcttc acactcgaag atttcgttgg ggactgggaa 60 cagacagccg cctacaacct ggaccaagtc cttgaacagg gaggtgtgtc cagtttgctg 120 cagaatctcg ccgtgtccgt aactccgatc caaaggattg tccggagcgg tgaaaatgcc 180 ctgaagatcg acatccatgt catcatcccg tatgaaggtc tgagcgccga ccaaatggcc 240 cagatcgaag aggtgtttaa ggtggtgtac cctgtggatg atcatcactt taaggtgatc 300 ctgccctatg gcacactggt aatcgacggg gttacgccga acatgctgaa ctatttcgga 360 cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg 420 tggaacggca acaaaattat cgacgagcgc ctgatcaccc ccgacggctc catgctgttc 480 cgagtaacca tcaacagttc gaattcgggt ggcggctctg gtggtggcag cggcggtggc 540 ggcggtggtg gcctgccgga aaccggtggc taagcggccg c 581 <210> 6 <211> 190 <212> PRT <213> Artificial Sequence <400> 6 Met His His His His His His Met Val Phe Thr Leu Glu Asp Phe Val 1 5 10 15 Gly Asp Trp Glu Gln Thr Ala Ala Tyr Asn Leu Asp Gln Val Leu Glu 20 25 30 Gln Gly Gly Val Ser Ser Leu Leu Gln Asn Leu Ala Val Ser Val Thr 35 40 45 Pro Ile Gln Arg Ile Val Arg Ser Gly Glu Asn Ala Leu Lys Ile Asp 50 55 60 Ile His Val Ile Ile Pro Tyr Glu Gly Leu Ser Ala Asp Gln Met Ala 65 70 75 80 Gln Ile Glu Glu Val Phe Lys Val Val Tyr Pro Val Asp Asp His His 85 90 95 Phe Lys Val Ile Leu Pro Tyr Gly Thr Leu Val Ile Asp Gly Val Thr 100 105 110 Pro Asn Met Leu Asn Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val 115 120 125 Phe Asp Gly Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn 130 135 140 Lys Ile Ile Asp Glu Arg Leu Ile Thr Pro Asp Gly Ser Met Leu Phe 145 150 155 160 Arg Val Thr Ile Asn Ser Ser Asn Ser Gly Gly Gly Ser Gly Gly Gly 165 170 175 Ser Gly Gly Gly Gly Gly Gly Gly Leu Pro Glu Thr Gly Gly 180 185 190 <210> 7 <211> 525 <212> DNA <213> Artificial Sequence <400> 7 atgcatcacc atcaccacca tatggtcttc acactcgaag atttcgttgg ggactgggaa 60 cagacagccg cctacaacct ggaccaagtc cttgaacagg gaggtgtgtc cagtttgctg 120 cagaatctcg ccgtgtccgt aactccgatc caaaggattg tccggagcgg tgaaaatgcc 180 ctgaagatcg acatccatgt catcatcccg tatgaaggtc tgagcgccga ccaaatggcc 240 cagatcgaag aggtgtttaa ggtggtgtac cctgtggatg atcatcactt taaggtgatc 300 ctgccctatg gcacactggt aatcgacggg gttacgccga acatgctgaa ctatttcgga 360 cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg 420 tggaacggca acaaaattat cgacgagcgc ctgatcaccc ccgacggctc catgctgttc 480 cgagtaacca tcaacagtgg cggtggcaac catgtgtaag aattc 525 <210> 8 <211> 172 <212> PRT <213> Artificial Sequence <400> 8 Met His His His His His His Met Val Phe Thr Leu Glu Asp Phe Val 1 5 10 15 Gly Asp Trp Glu Gln Thr Ala Ala Tyr Asn Leu Asp Gln Val Leu Glu 20 25 30 Gln Gly Gly Val Ser Ser Leu Leu Gln Asn Leu Ala Val Ser Val Thr 35 40 45 Pro Ile Gln Arg Ile Val Arg Ser Gly Glu Asn Ala Leu Lys Ile Asp 50 55 60 Ile His Val Ile Ile Pro Tyr Glu Gly Leu Ser Ala Asp Gln Met Ala 65 70 75 80 Gln Ile Glu Glu Val Phe Lys Val Val Tyr Pro Val Asp Asp His His 85 90 95 Phe Lys Val Ile Leu Pro Tyr Gly Thr Leu Val Ile Asp Gly Val Thr 100 105 110 Pro Asn Met Leu Asn Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val 115 120 125 Phe Asp Gly Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn 130 135 140 Lys Ile Ile Asp Glu Arg Leu Ile Thr Pro Asp Gly Ser Met Leu Phe 145 150 155 160 Arg Val Thr Ile Asn Ser Gly Gly Gly Asn His Val 165 170 <210> 9 <211> 525 <212> DNA <213> Artificial Sequence <400> 9 atgcatcacc atcaccacca tatggtcttc acactcgaag atttcgttgg ggactgggaa 60 cagacagccg cctacaacct ggaccaagtc cttgaacagg gaggtgtgtc cagtttgctg 120 cagaatctcg ccgtgtccgt aactccgatc caaaggattg tccggagcgg tgaaaatgcc 180 ctgaagatcg acatccatgt catcatcccg tatgaaggtc tgagcgccga ccaaatggcc 240 cagatcgaag aggtgtttaa ggtggtgtac cctgtggatg atcatcactt taaggtgatc 300 ctgccctatg gcacactggt aatcgacggg gttacgccga acatgctgaa ctatttcgga 360 cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg 420 tggaacggca acaaaattat cgacgagcgc ctgatcaccc ccgacggctc catgctgttc 480 cgagtaacca tcaacagtgg cggtggcccc attcagtaag aattc 525 <210> 10 <211> 172 <212> PRT <213> Artificial Sequence <400> 10 Met His His His His His His Met Val Phe Thr Leu Glu Asp Phe Val 1 5 10 15 Gly Asp Trp Glu Gln Thr Ala Ala Tyr Asn Leu Asp Gln Val Leu Glu 20 25 30 Gln Gly Gly Val Ser Ser Leu Leu Gln Asn Leu Ala Val Ser Val Thr 35 40 45 Pro Ile Gln Arg Ile Val Arg Ser Gly Glu Asn Ala Leu Lys Ile Asp 50 55 60 Ile His Val Ile Ile Pro Tyr Glu Gly Leu Ser Ala Asp Gln Met Ala 65 70 75 80 Gln Ile Glu Glu Val Phe Lys Val Val Tyr Pro Val Asp Asp His His 85 90 95 Phe Lys Val Ile Leu Pro Tyr Gly Thr Leu Val Ile Asp Gly Val Thr 100 105 110 Pro Asn Met Leu Asn Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val 115 120 125 Phe Asp Gly Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn 130 135 140 Lys Ile Ile Asp Glu Arg Leu Ile Thr Pro Asp Gly Ser Met Leu Phe 145 150 155 160 Arg Val Thr Ile Asn Ser Gly Gly Gly Pro Ile Gln 165 170 <210> 11 <211> 620 <212> DNA <213> Artificial Sequence <400> 11 atgcatcacc atcaccacca tatggtcttc acactcgaag atttcgttgg ggactgggaa 60 cagacagccg cctacaacct ggaccaagtc cttgaacagg gaggtgtgtc cagtttgctg 120 cagaatctcg ccgtgtccgt aactccgatc caaaggattg tccggagcgg tgaaaatgcc 180 ctgaagatcg acatccatgt catcatcccg tatgaaggtc tgagcgccga ccaaatggcc 240 cagatcgaag aggtgtttaa ggtggtgtac cctgtggatg atcatcactt taaggtgatc 300 ctgccctatg gcacactggt aatcgacggg gttacgccga acatgctgaa ctatttcgga 360 cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg 420 tggaacggca acaaaattat cgacgagcgc ctgatcaccc ccgacggctc catgctgttc 480 cgagtaacca tcaacagttc gaattcgggt ggcggctctg gtggtggcag cggcggtggc 540 ggcggtggtg gcctgccgga aaccggtggc ggtggcgtga ccggctaccg tctgttcgaa 600 gagattctgt aagcggccgc 620 <210> 12 <211> 203 <212> PRT <213> Artificial Sequence <400> 12 Met His His His His His His Met Val Phe Thr Leu Glu Asp Phe Val 1 5 10 15 Gly Asp Trp Glu Gln Thr Ala Ala Tyr Asn Leu Asp Gln Val Leu Glu 20 25 30 Gln Gly Gly Val Ser Ser Leu Leu Gln Asn Leu Ala Val Ser Val Thr 35 40 45 Pro Ile Gln Arg Ile Val Arg Ser Gly Glu Asn Ala Leu Lys Ile Asp 50 55 60 Ile His Val Ile Ile Pro Tyr Glu Gly Leu Ser Ala Asp Gln Met Ala 65 70 75 80 Gln Ile Glu Glu Val Phe Lys Val Val Tyr Pro Val Asp Asp His His 85 90 95 Phe Lys Val Ile Leu Pro Tyr Gly Thr Leu Val Ile Asp Gly Val Thr 100 105 110 Pro Asn Met Leu Asn Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val 115 120 125 Phe Asp Gly Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn 130 135 140 Lys Ile Ile Asp Glu Arg Leu Ile Thr Pro Asp Gly Ser Met Leu Phe 145 150 155 160 Arg Val Thr Ile Asn Ser Ser Asn Ser Gly Gly Gly Ser Gly Gly Gly 165 170 175 Ser Gly Gly Gly Gly Gly Gly Gly Leu Pro Glu Thr Gly Gly Gly Gly 180 185 190 Val Thr Gly Tyr Arg Leu Phe Glu Glu Ile Leu 195 200 <210> 13 <211> 566 <212> DNA <213> Artificial Sequence <400> 13 atgcatcacc atcaccacca tatggtcttc acactcgaag atttcgttgg ggactgggaa 60 cagacagccg cctacaacct ggaccaagtc cttgaacagg gaggtgtgtc cagtttgctg 120 cagaatctcg ccgtgtccgt aactccgatc caaaggattg tccggagcgg tgaaaatgcc 180 ctgaagatcg acatccatgt catcatcccg tatgaaggtc tgagcgccga ccaaatggcc 240 cagatcgaag aggtgtttaa ggtggtgtac cctgtggatg atcatcactt taaggtgatc 300 ctgccctatg gcacactggt aatcgacggg gttacgccga acatgctgaa ctatttcgga 360 cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac agggaccctg 420 tggaacggca acaaaattat cgacgagcgc ctgatcaccc ccgacggctc catgctgttc 480 cgagtaacca tcaacagtgg cggtggcaac ggtattggtg gcgtgaccgg ctaccgtctg 540 ttcgaagaga ttctgtaagc ggccgc 566 <210> 14 <211> 185 <212> PRT <213> Artificial Sequence <400> 14 Met His His His His His His Met Val Phe Thr Leu Glu Asp Phe Val 1 5 10 15 Gly Asp Trp Glu Gln Thr Ala Ala Tyr Asn Leu Asp Gln Val Leu Glu 20 25 30 Gln Gly Gly Val Ser Ser Leu Leu Gln Asn Leu Ala Val Ser Val Thr 35 40 45 Pro Ile Gln Arg Ile Val Arg Ser Gly Glu Asn Ala Leu Lys Ile Asp 50 55 60 Ile His Val Ile Ile Pro Tyr Glu Gly Leu Ser Ala Asp Gln Met Ala 65 70 75 80 Gln Ile Glu Glu Val Phe Lys Val Val Tyr Pro Val Asp Asp His His 85 90 95 Phe Lys Val Ile Leu Pro Tyr Gly Thr Leu Val Ile Asp Gly Val Thr 100 105 110 Pro Asn Met Leu Asn Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val 115 120 125 Phe Asp Gly Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn 130 135 140 Lys Ile Ile Asp Glu Arg Leu Ile Thr Pro Asp Gly Ser Met Leu Phe 145 150 155 160 Arg Val Thr Ile Asn Ser Gly Gly Gly Asn Gly Ile Gly Gly Val Thr 165 170 175 Gly Tyr Arg Leu Phe Glu Glu Ile Leu 180 185 <210> 15 <211> 15 <212> PRT <213> Artificial Sequence <400> 15 Gly Gly Gly Gly Val Thr Gly Tyr Arg Leu Phe Glu Glu Ile Leu 1 5 10 15 <210> 16 <211> 15 <212> PRT <213> Artificial Sequence <400> 16 Gly Ile Gly Gly Val Thr Gly Tyr Arg Leu Phe Glu Glu Ile Leu 1 5 10 15 <210> 17 <211> 15 <212> PRT <213> Artificial Sequence <400> 17 Phe Gly Gly Gly Val Thr Gly Tyr Arg Leu Phe Glu Glu Ile Leu 1 5 10 15

Claims

1. A composition for determining the activity of a protein ligase, comprising a corresponding ligation-type LgBiT protein and a ligation-type SmBiT; the ligation-type LgBiT protein is characterized in that an amino acid sequence recognized by the protein ligase to be detected is fused to the C-terminus of LgBiT; the ligation-type SmBiT is characterized in that a nucleophilic attack sequence recognized by the protein ligase to be detected is introduced to the N-terminus of SmBiT; When the protein ligase to be tested is Sortase A ligase, the ligation-type LgBiT protein is LgBiT-long-LPETG, and its amino acid sequence is shown in Seq ID NO.6; the ligation-type SmBiT is 4G-SmBiT, and its amino acid sequence is shown in Seq ID NO.15; When the protein ligase to be tested is an asparagine protease-type protein ligase, the ligation-type LgBiT protein is LgBiT-NHV, and its amino acid sequence is shown in Seq ID NO.8; the ligation-type SmBiT is GI-SmBiT, and its amino acid sequence is shown in Seq ID NO.16; When the protein ligase to be tested is a proline protease-type protein ligase, the LgBiT ligase protein is LgBiT-PIQ, and its amino acid sequence is shown in Seq ID NO.10; the ligase SmBiT is FG-SmBiT, and its amino acid sequence is shown in Seq ID NO.

17.

2. A method for determining the activity of various protein ligases based on bioluminescence, characterized in that: The method using the composition according to claim 1 is a method for non-disease diagnosis and treatment purposes.

3. The method according to claim 2, characterized in that The method comprises the steps of mixing a dilution of the composition according to claim 1 with a protein ligase to be tested to react, and performing bioluminescence measurement on the reaction solution.

4. Use of the composition according to claim 1 in any of the following: 1) Application in the preparation of products for determining protein ligase activity; 2) Application in determining protein ligase activity; 3) Application in screening protein ligases from plants or microorganisms; 4) Application in identifying protein ligases from plants or microorganisms; 5) Application in screening of protein ligase inhibitors; The application is for purposes other than disease diagnosis and treatment.

5. The method according to any one of claims 2 to 3, used in any of the following: 1) Application in screening protein ligases from plants or microorganisms; 2) Application in identifying protein ligases from plants or microorganisms; 3) Application in screening of protein ligase inhibitors; The application is for purposes other than disease diagnosis and treatment.

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