CN116042766A - Composition, method and application for measuring protein ligase activity based on bioluminescence - Google Patents

Composition, method and application for measuring protein ligase activity based on bioluminescence Download PDF

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CN116042766A
CN116042766A CN202210120982.2A CN202210120982A CN116042766A CN 116042766 A CN116042766 A CN 116042766A CN 202210120982 A CN202210120982 A CN 202210120982A CN 116042766 A CN116042766 A CN 116042766A
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smbit
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郭占云
张聪慧
邵晓霞
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Tongji University
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Abstract

The invention relates to a novel method for measuring the activity of protein ligase based on bioluminescence, which is universally applicable to various protein ligases, can be used for 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 invention is characterized in that a recognition sequence of a protein ligase is fused at the C-terminal end of a nonactive NanoLuc luciferase large fragment (LgBiT) by recombinant expression or other methods, and a nucleophilic attack sequence recognized by the protein ligase is introduced at the N-terminal end of a SmBiT complementary tag with low affinity by chemical synthesis or other methods. The activity determination method is based on bioluminescence detection, has the advantages of high sensitivity, simplicity and convenience in operation, protease interference resistance, suitability for high-throughput screening and the like, and plays an important role in researching known protein ligase or screening unknown protein ligase.

Description

Composition, method and application for measuring protein ligase activity based on bioluminescence
Technical Field
The invention relates to biological detection, enzymology and genetic engineering technology, in particular to a novel method for measuring the activity of various protein ligase based on bioluminescence, which comprises key design, components, steps and various applications of the method.
Background
Protein ligases (or polypeptide ligases) are a special class of proteases that can link two peptide chains together by peptide bonds or form a cyclic peptide from one peptide chain by peptide bonds via a transpeptidation mechanism. More protein ligases are currently being studied, including gram positive bacterial-derived sortase (sortase) and some plant-derived asparaginase (asparaginyl endopeptidase, abbreviated as AEP) protein ligases, such as buttealase-1 from pterocele (Clitoria ternatea) and OaAEP1 from blue-flower otoweed (Oldenlandia affinis), among others. The protein ligase is widely used for modification of proteins and polypeptides and cyclopeptide synthesis, has important application and development values, therefore, the activity characterization of the known protein ligase is required, and various novel protein ligases are continuously screened and identified so as to obtain the protein ligase with better and more practical performances. The existing methods for measuring the activity of the protein ligase are generally based on fluorescence detection (fluorescent probe labeling substrate), high performance liquid chromatography detection or mass spectrometry detection, and all the methods have certain defects such as low sensitivity, long time consumption, easy interference by protease, expensive instrument and equipment requirement, difficulty in high-throughput screening and the like.
Disclosure of Invention
The invention aims at: 1) Various connection type LgBiT and connection type SmBiT for measuring the activity of protein ligase are provided, and the design key points and the preparation method thereof are provided. 2) A method for measuring the activity of various protein ligases based on bioluminescence by using a connected LgBiT and a connected SmBiT is provided. 3) Various uses of the activity assay are provided, including screening for various unknown protein ligases from plants or other sources, and characterizing various known protein ligases.
The invention provides a composition, which comprises a connection type LgBiT and a connection type SmBiT, wherein the connection type LgBiT is a protein obtained by fusing a recognition sequence of a protein ligase to be detected at the C-terminal of the LgBiT through a section of connection sequence; the connecting SmBiT is a polypeptide obtained by introducing a nucleophilic attack sequence recognized by a protein ligase to be detected into the N-end of the SmBiT.
The background luciferase activity of the connected LgBiT is low, but the background luciferase activity and the luciferase activity after the complementary HiBiT can be recovered, which are close to the corresponding values of the original LgBiT, generally speaking, at least the background luciferase activity of the connected LgBiT is less than 1% of the high luciferase activity after the complementary HiBiT, if the background luciferase activity of the connected LgBiT is less than one thousandth or one thousandth of the high luciferase activity after the complementary HiBiT, the effect is better.
When the protein ligase to be detected is Sortase A, the connection type LgBiT is LgBiT-long-LPETG, and the amino acid sequence of the connection type LgBiT-long-LPETG is shown as the sequence of Seq ID No. 6; the connecting SmBiT is 4G-SmBiT, and the amino acid sequence of the connecting SmBiT is shown as Seq ID No. 15.
When the protein ligase to be detected is asparagine protease type protein ligase, the connection type LgBiT is LgBiT-NHV, and the amino acid sequence of the connection type LgBiT-NHV is shown as the sequence of Seq ID No. 8; the connecting SmBiT is GI-SmBiT, and the amino acid sequence of the connecting SmBiT is shown as Seq ID No. 16.
When the protein ligase to be detected is prolyl protease type protein ligase, the connection type LgBiT is LgBiT-PIQ, and the amino acid sequence of the connection type LgBiT-PIQ is shown as the sequence of Seq ID No. 10; the connecting SmBiT is FG-SmBiT, and the amino acid sequence is shown as Seq ID No. 17.
A method for measuring the activity of various protein ligases based on bioluminescence, which uses the composition.
The method comprises the steps of mixing the composition with protein ligase to be detected to enable the protein ligase to be detected to carry out connection reaction at a higher concentration (mu mol/l level), then properly diluting the reaction mixture to reduce the background activity caused by complementation of the connection type LgBiT and the connection type SmBiT bimolecular, and finally carrying out bioluminescence measurement on the diluted reaction solution.
The use of the composition or the method in any of the following is also within the scope of the invention:
1) The application in preparing the product for measuring the activity of the protein ligase;
2) The application in the determination of protein ligase activity;
3) Use in screening protein ligases of plant, animal or microbial origin;
4) Use in the identification of protein ligases of plant, animal or microbial origin;
the protein ligase is various protein ligases such as AEP type protein ligase, PEP type protein ligase, sortase and the like.
The invention provides an application of the method for measuring the activity of protein ligase in the characterization of the protein ligase; application in the activity tracking of protein ligase; use in a protein ligase inhibitor screen.
The invention provides a novel method for measuring the activity of protein ligase based on bioluminescence, which is universally applicable to various protein ligases, can be used for 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 invention is characterized in that a recognition sequence of a protein ligase is fused at the C-terminal end of a nonactive NanoLuc luciferase large fragment (LgBiT) by recombinant expression or other methods, and a nucleophilic attack sequence recognized by the protein ligase is introduced at the N-terminal end of a SmBiT complementary tag with low affinity by chemical synthesis or other methods. The activity determination method is based on bioluminescence detection, has the advantages of high sensitivity, simplicity and convenience in operation, protease interference resistance, suitability for high-throughput screening and the like, and plays an important role in researching known protein ligase or screening unknown protein ligase.
Drawings
Fig. 1: the principle and the operation flow of the novel method for measuring the activity of the protein ligase based on bioluminescence. (A) A universal form of ligbit and ligo SmBiT for protein ligase activity assay. A suitable recognition sequence of the connecting peptide fusion protein ligase is adopted at the C-end of the LgBiT, and a nucleophilic attack sequence recognized by the protein ligase is introduced at the N-end of the SmBiT. (B) General procedure for the novel method for determining protein ligase activity based on bioluminescence. The ligation reaction is carried out under the catalysis of protein ligase on the high-concentration ligation LgBiT and ligation SmBiT, a small amount of reaction mixture (such as 10 mu l) is taken out for proper dilution (usually 100 times to 100000 times) at different reaction times, and then a small amount of diluted sample (such as 10 mu l) is taken and added to the NanoLuc substrate, and then bioluminescence is measured on an enzyme-labeled instrument.
Fig. 2: the new method for determining protein ligase activity based on bioluminescence was validated with bacterial Sortase a. (A) Schematic of the ligation type LgBiT and SmBiT and the expected ligation products of both for Sortase a ligase activity assay. Wherein amino acids are indicated by single letters. (B) The luciferase activity and purity of the recombinantly expressed ligation-type LgBiT and the expected ligation product were identified by bioluminescence and SDS-PAGE. (C) The recombinant annular Sortase A catalyzes the change of bioluminescence along with the reaction time in the connection process of LgBiT-long-LPETG and 4G-SmBiT. Samples (10. Mu.l) were taken at different reaction times and diluted 10000-fold in two steps, and then 10. Mu.l of the diluted sample was taken to measure bioluminescence. (D, E) Sortase A catalyzed ligation of LgBiT-long-LPETG and 4G-SmBiT was detected by bioluminescence and SDS-PAGE. For measuring bioluminescence, samples (10. Mu.l) were taken at different reaction times and diluted 100000 times in three steps, and then 10. Mu.l of the diluted sample was taken for measuring bioluminescence. For SDS-PAGE detection samples (10. Mu.l) were taken at different reaction times, mixed with 40. Mu.l SDS-PAGE buffer, boiled to 10. Mu.l loading, electrophoresed and stained with Coomassie blue. The LgBiT-long-LPETG bands are indicated by asterisks, the ligation bands are indicated by well marks, and the ligation bands coincide with the recombinant circular Sortase A bands.
Fig. 3: a novel method for determining the activity of protein ligase based on bioluminescence was verified by using plant Butelase-1. (A) Schematic of the ligation type LgBiT and SmBiT and the expected ligation products of the two for Butelase-1 ligase activity assay. Wherein amino acids are indicated by single letters. (B) The luciferase activity and purity of the recombinantly expressed ligation-type LgBiT and the expected ligation products were identified by bioluminescence and SDS-PAGE. (C) The crude extract of pteridopsis japonica catalyzes the change of bioluminescence during the process of connecting LgBiT-NHV with GI-SmBiT. Samples (10. Mu.l) were taken at different reaction times and diluted 3000 times in two steps, and then 10. Mu.l of the diluted sample was taken to determine bioluminescence. (D) The partially purified Butelase-1 catalyzes the ligation of LgBiT-NHV and GI-SmBiT. Samples (10. Mu.l) were taken at different reaction times and diluted 10000 times in two steps, then 10. Mu.l 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. (E) Butelase-1 catalyzed ligation of LgBiT-NHV and GI-SmBiT was partially purified by bioluminescence and SDS-PAGE detection. When measuring bioluminescence, samples (10. Mu.l) were taken at different reaction times and diluted 100000 times in three steps, then 10. Mu.l of the diluted sample was taken to measure bioluminescence, and then the concentration of the ligation product in the reaction mixture was calculated based on the ratio of ligation products. For SDS-PAGE detection samples (10. Mu.l) were taken at different reaction times, mixed with 20. Mu.l SDS-PAGE buffer, boiled to 10. Mu.l loading, electrophoresed and stained with Coomassie blue. Ligation product bands are indicated by asterisks.
Fig. 4: plants likely to express AEP type protein ligase were selected using LgBiT-NHV and GI-SmBiT. Grinding plant tissues in PBS and centrifuging to obtain supernatant, namely crude extract. The crude extract was mixed with LgBiT-NHV and GI-SmBiT in the indicated proportions, incubated at 25℃and sampled at different reaction times (10. Mu.l), diluted 3000 times in two steps and the bioluminescence was determined by taking 10. Mu.l of the diluted sample. The photographs show the plant parts used to prepare the crude ligase extract.
Fig. 5: plants likely to express PEP-type protein ligase were selected with LgBiT-PIQ and FG-SmBiT. (A) Amino acid sequences of cyclic peptide precursors having Pro residues at the C-terminus of some mature peptides. Wherein the mature peptide portion of the cyclic peptide is represented by a red color. (B) Design of the connection type LgBiT and SmBiT for screening PEP type protein ligase. Wherein amino acids are indicated by single letters. And (C-F) measuring the activity of PEP type protein ligase in the plant crude extract. Grinding plant tissues in PBS and centrifuging to obtain supernatant, namely crude extract. The crude extract was mixed with LgBiT-PIQ and FG-SmBiT in the indicated proportions, incubated at 25℃and sampled at different reaction times (10. Mu.l), diluted 2000-fold in two steps and the bioluminescence was determined by taking 10. Mu.l of the diluted sample. The photographs show the plant parts used to prepare the crude ligase extract.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. Unless otherwise indicated, it may be carried out according to the methods set forth in the laboratory manuals of "molecular cloning laboratory Manual (third edition) (Cold Spring Harbor laboratory Press)," cell laboratory manual "(scientific Press, beijing, china, 2001), etc., familiar to those skilled in the art.
pET expression vectors in the examples described below, disclosed in the documents 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; coli expression strain BL21 (DE 3) is disclosed in the documents 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 is available from the university of ataxia for use in validating the present invention.
The 80 plants in the following examples were all from the inventors' own collection, wherein, the pteris beans were collected in southwest of China for 2021, 3 months; other plant samples were collected in Shanghai, china, between 7 and 8 months of 2021.
Bioluminescence (Bioluminescence) refers to a type of chemiluminescent reaction catalyzed by various luciferases, and has the advantages of low detection background, wide linear range, strong anti-interference capability and the like, and is widely used for various quantitative detection. Typical luciferases used in bioluminescence assays include firefly luciferases, renilla luciferases, nanoLuc luciferases developed in recent years, and the like. The NanoLuc luciferase has the advantages of small component (171 amino acids), stable structure, high luminous efficiency and the like. Based on NanoLuc, promega has recently developed NanoLuc bimolecular interaction technology (NanoLuc Binary Technology, abbreviated NanoBiT) which comprises a large fragment of nonactive NanoLuc (abbreviated LgBiT, consisting of 159 amino acid residues) and two complementary tags, namely a low affinity SmBiT tag and a high affinity HiBiT tag, both of which have only 11 amino acid residues. Low affinity LgBiT and SmBiT systems are commonly used to study the dynamic interactions of two protein molecules, while high affinity LgBiT and HiBiT systems can be used to quantify HiBiT tag fused proteins or other molecules.
Due to the low affinity of both (K) d The value is 190 mu mol/l), smBiT can realize bimolecular complementation with LgBiT at high concentration, so that the luciferase activity is recovered. It is speculated that if SmBiT can be covalently linked to the C-terminus of LgBiT, the linked SmBiT not only can effectively restore the activity of LgBiT, but also is not affected by free SmBiT. Based on this, the present invention has developed a novel method for determining the activity of various protein ligases by using LgBiT and SmBiT, i.e. by fusing the recognition sequence of a protein ligase or a class of protein ligases at the C-terminus of LgBiT via a linker sequence and introducing a nucleophilic attack sequence recognized by this protein ligase or such class of protein ligases at the N-terminus of SmBiT (fig. 1A). If the LgBiT and SmBiT can be linked together by expected protein ligase, the linked product can restore the activity of luciferase, and after the NanoLuc substrate is added, the quantitative determination can be carried out by bioluminescence, and the detection limit can reach the fmol level or even lower.
The invention provides a connection type LgBiT and a connection type SmBiT as well as design key points and a preparation method thereof.
The design key point of the connection type LgBiT and the preparation method are as follows: the recognition sequence of a known protein ligase or a putative protein ligase or a recognition sequence of a class of proteins is fused to the C-terminus of the original LgBiT protein by a suitable linker peptide (FIG. 1A). By changing the length or amino acid composition of the connecting peptide, the background luciferase activity of the connecting LgBiT is ensured to be low, and the HiBiT can recover high luciferase activity after complementation. For example, by introducing a known Sortase a recognition sequence and optimizing its linker peptide in the present invention, lgBiT-long-LPETG of low background luciferase activity for Sortase a ligase activity assay was obtained; by introducing a known Butelase-1 recognition sequence, lgBiT-NHV for measuring Butelase-1 protein ligase activity and screening asparagine protease (AEP) protein ligase is obtained; lgBiT-PIQ for screening PEP type protein ligases was obtained by introducing a putative recognition sequence for a protease of the type prolyl-amidase (PEP) protein ligase. Other researchers can easily design the connection type LgBiT for measuring the activity of various protein ligases according to the design key points provided by the patent.
The invention establishes a method for preparing various connection type LgBiT by utilizing escherichia coli recombinant expression, optimizes the induction conditions including the induction temperature and the concentration of the inducer. To facilitate purification of various ligation type LgBiT, a 6XHis tag is fused at its N-terminus, so that the recombinantly expressed LgBiT can be purified by solid phase metal chelate chromatography (Ni 2+ Column) purification. Then, through one step of anion exchange chromatography, various connection type LgBiT of single strip can be obtained. To determine whether the designed ligation LgBiT is available, it is necessary to determine its background luciferase activity and its complementary HiBiT luciferase activity.
The design key points of the connection type SmBiT and the preparation method are as follows: a suitable linker may be added between the nucleophilic attack sequence and the SmBiT sequence by introducing a nucleophilic attack sequence recognized by a protein ligase or classes or a putative nucleophilic attack sequence of a protein ligase or classes at the N-terminus of the original SmBiT (fig. 1A). For example, four consecutive Gly residues are introduced as affinity attack sequences recognized by the Sortase A in the invention, so as to obtain 4G-SmBiT for measuring the activity of the Sortase A; by introducing Gly-Ile as an affinity attack sequence recognized by Butelase-1 and adding two Glys as connectors, the GI-SmBiT for measuring Butelase-1 activity and screening AEP protein ligase is obtained; FG-SmBiT for screening PEP type protein ligase was obtained by introducing Phe as an affinity attack sequence recognized by PEP type protein ligase and adding three Glys as linkers. Other researchers can easily design the connection type SmBiT for measuring the activity of various protein ligases according to the design key points provided by the patent.
The various linked smbits can be chemically synthesized by a mature solid phase polypeptide synthesis process, then purified by High Performance Liquid Chromatography (HPLC) using a C18 reverse phase column, and confirmed by mass spectrometry to determine molecular weight.
The invention provides a method for measuring the activity of various protein ligases based on bioluminescence by using a connection type LgBiT and a connection type SmBiT.
In order to conduct the activity measurement of various protein ligases by using the ligation type LgBiT and the ligation type SmBiT, a simple and reasonable operation procedure, namely three steps of ligation reaction, dilution of a ligation reaction mixture and bioluminescence measurement, was developed (FIG. 1B). Protein ligases typically require higher substrate concentrations to effectively catalyze ligation reactions, and therefore higher concentrations (in the order of μmol/l) of ligation LgBiT and ligation SmBiT are used in ligation reactions. However, lgBiT and SmBiT can undergo bimolecular complementation at high concentrations, resulting in strong background luminescence, resulting in failure to measure small amounts of ligation products (table 1). One key strategy for success of this method is to properly dilute the ligation reaction mixture (FIG. 1B), where the background luminescence generated by the complementary LgBiT and SmBiT bimolecular reaction decreases as the square of the dilution factor, while the signal luminescence generated by the ligation product decreases linearly as the dilution factor (Table 1), so that the luminescence generated by the ligation product is significantly greater than the background luminescence under the appropriate dilution conditions. Under appropriate dilution conditions, ligation products were detected very sensitively even if only one thousandth of the ligation LgBiT was catalyzed by the protein ligase to ligation to the ligation type SmBiT (Table 1). According to the three experimental steps designed, the activity of various known or unknown protein ligases can be determined using the corresponding ligbit and ligit.
Table 1 shows the background luminescence and the ligation yield of LgBiT and SmBiT bimolecular complementation under different dilution conditions obtained by theoretical calculationThe signal generated by the substance emits light. Assume that the K bound by the LgBiT and SmBiT of the ligation type d Value K combined with original LgBiT and original SmBiT d The values are identical, i.e. 190. Mu. Mol/l. It is assumed that 1% or 0.1% of the ligation LgBiT and SmBiT are linked together by a proteolytic ligase in the ligation reaction. The specific activities of the luciferase enzymes of the ligation products and SmBiT complementary LgBiT are 5×10 5 RLU/fmol (measured on an iD3 microplate reader using 384 well plates). After the ligation reaction was initiated, samples were taken at various reaction times and diluted appropriately, and then diluted samples were taken into 384-well plates (10. Mu.l/well). Bioluminescence was measured on a microplate reader immediately after addition of diluted NanoLuc luciferase substrate (10 μl/well). Red indicates that the signal luminescence is significantly higher than the background luminescence in this diluted condition.
Table 1: theoretical calculation shows that LgBiT and SmBiT bimolecular complementation produces background luminescence and signal luminescence produced by connection products under different dilution conditions
Figure BDA0003498394940000071
The invention provides an application of a method for measuring the activity of protein ligase based on bioluminescence.
The method for measuring the protein ligase activity based on bioluminescence has the advantages of high sensitivity, simple operation, protease interference resistance, suitability for high-throughput screening and the like, and has wide application prospects in protein ligase research, including screening and identifying various unknown protein ligases from plants or other sources and related research of various known protein ligases. For example, by screening crude extracts of 80 common plants, 5 of them were identified, including zephyranthes candida (Zephyranthes candida), hibiscus (Hibiscus syriacus), sparrow (Angelonia angustifolia), hosta plantaginea (Hosta plantaginea) and winter jasmine (Jasminum nudiflorum), likely to express AEP-type protein ligase; among them, 4 plants including sparrow (Angelonia angustifolia), flower She Qingmu (Aucuba japonica var. Variegate), herba Eupatorii (Eupatorium fortunei) and Duck grass (Linderniantipta) are possible to express PEP protein ligase. By using the activity determination method, a large number of plants, animals and microorganisms can be rapidly screened at low cost, and various types of protein ligases can be identified. In addition, the activity determination method can be used for various researches such as characterization, activity tracking, inhibitor screening and the like of known protein ligase.
Example 1 verification of novel methods for protein ligase Activity based on bioluminescence Using bacterial Sortase A
1. Design of ligation type LgBiT and SmBiT for determination of Sortase a ligase activity:
the recognition sequence of Sortase a is known to consist of 5 amino acid residues, usually LPETG, and is therefore introduced at the C-terminus of LgBiT and protected by adding an extra Gly thereto (fig. 2A). Meanwhile, connecting peptides with different lengths are tried to be introduced between LgBiT and a recognition sequence, so that the background luciferase activity of the obtained connecting LgBiT is low, but the luciferase activity can be recovered after the connecting LgBiT is complementary with HiBiT. When the connecting peptide is 3 Gly residues, the obtained connecting LgBiT is named as LgBiT-LPETG, and the amino acid sequence of the connecting peptide is shown as Seq ID No. 4; when the linker peptide was 18 amino acid residues, it was designated LgBiT-long-LPETG, and its amino acid sequence was as shown in Seq ID No. 6.
The nucleophilic attack sequence recognized by Sortase a is typically a plurality of consecutive Gly residues, thus introducing 4 consecutive Gly residues at the N-terminus of SmBiT, designated 4G-SmBiT (fig. 2A), the sequence of which is shown in Seq ID No. 16.
In addition, the expected ligation products of LgBiT-long-LPETG and 4G-SmBiT were also designed and named LgBiT-long-LPET-4G-SmBiT, the amino acid sequence of which is shown in Seq ID No.12, to verify if the expected ligation products had luciferase activity (FIG. 2A).
2. Chemical synthesis and purification of 4G-SmBiT:
the 4G-SmBiT is chemically synthesized by a conventional solid-phase peptide synthesis method, and the synthesized crude peptide is purified by a High Performance Liquid Chromatography (HPLC) with a C18 reverse phase column to obtain a pure sample, and mass spectrum shows that the molecular weight is correct. After purification, the lyophilized 4G-SmBiT sample was dissolved in 1.0mmol/l of diluted hydrochloric acid (pH 3.0), and the concentration was determined by 280nm ultraviolet light absorption (extinction coefficient: 1490 l/mol/cm), and then split-packed (20-50. Mu.l/tube), frozen at-80℃for the subsequent experiments.
2. Recombinant expression, separation and purification of various LgBiT proteins and fusion proteins thereof:
1. preparation of recombinant vectors
1.1 replacing the sequence between NdeI and EcoRI sites in pET expression vector with the coding sequence of chemically synthesized LgBiT (shown as sequence Seq ID No. 1), and keeping other sequences unchanged, the obtained recombinant vector is pET/LgBiT, which codes an LgBiT protein with 6xHis tag at N-terminal (shown as sequence Seq ID No. 2).
1.2 the LgBiT coding sequence in step 1.1 is replaced by LgBiT-LPETG (shown as sequence Seq ID No. 3), and the other steps are unchanged, so that a recombinant vector pET/LgBiT-LPETG is obtained, and the LgBiT-LPETG protein is coded (shown as sequence Seq ID No. 4).
1.3 replacing the LgBiT coding sequence in the step 1.1 with LgBiT-long-LPETG (shown as sequence Seq ID No. 5), and obtaining a recombinant vector pET/LgBiT-long-LPETG with the coding LgBiT-long-LPETG protein (shown as sequence Seq ID No. 6) without changing other steps.
1.4 the LgBiT coding sequence in the step 1.1 is replaced by LgBiT-long-LPET-4G-SmBiT (shown as sequence Seq ID No. 13), and other steps are unchanged, so that a recombinant vector pET/LgBiT-long-LPET-4G-SmBiT is obtained, and the LgBiT-long-LPET-4G-SmBiT protein is coded (shown as sequence Seq ID No. 14).
The above LgBiT-LPETG, lgBiT-long-LPETG and LgBiT-long-LPET-4G-SmBiT coding sequences can be obtained by PCR amplification or chemical synthesis. The coding sequences of the various LgBiT were verified by DNA sequencing.
2. Preparation and purification of proteins
2.1 Preparation of LgBiT protein
Transforming the recombinant expression vector pET/LgBiT into an escherichia coli expression strain BL21 (DE 3) to obtain a transformed strain BL21 (DE 3) -pET/LgBiT; the transformed strain BL21 (DE 3) -pET/LgBiT was shake-cultured in LB liquid medium (containing 100mg/l ampicillin) in a shaker at 37℃until the optical density OD600 reached 1-2. Then the culture solution is cooled to 16 ℃, and thenAdding inducer IPTG to 0.5mmol/l, and continuously shaking culture at 16 deg.C overnight. Then, the cells were collected by centrifugation (5000 g,10 min) and resuspended in an appropriate amount of lysis buffer (20 mmol/l phosphate, pH7.4,0.5mol/l NaCl) and lysed by sonication. Then centrifuged (12000 g,20 min), the supernatant was taken and passed through Ni 2+ And (5) purifying by a column. LgBiT protein was eluted with 250mmol/l imidazole (in lysis buffer), the eluted fractions were collected and dialyzed against 10mmol/l Tris-Cl (pH 7.5) overnight. After dialysis, centrifugation (10000 g,10 min) and purification of the supernatant by EDAE ion exchange chromatography, linear gradient elution with NaCl, eluent A of 10mmol/l Tris-Cl (pH 7.5) and eluent B of 500mmol/l NaCl were added to eluent A. LgBiT protein elution peaks were collected, identified as single bands by SDS-PAGE, and the size was expected (FIG. 2B). The purified LgBiT is quantified through 280nm ultraviolet light absorption (the extinction coefficient of the LgBiT is 20000 l/mol/cm), and then split charging (20-50 mu l/tube) is carried out, so that LgBiT protein is obtained, and the LgBiT protein is frozen at-80 ℃ for subsequent experiments.
2.2 Preparation of LgBiT-LPETG protein
And (3) replacing the recombinant expression vector pET/LgBiT in the step (2.1) with pET/LgBiT-LPETG, and obtaining a transformed strain BL21 (DE 3) -pET/LgBiT-LPETG without changing other steps, thereby further obtaining LgBiT-LPETG protein.
2.3 Preparation of LgBiT-long-LPETG protein
And (3) replacing the recombinant expression vector pET/LgBiT in the step (2.1) with pET/LgBiT-long-LPETG, and obtaining a transformed strain BL21 (DE 3) -pET/LgBiT-long-LPETG without changing other steps, thereby further obtaining LgBiT-long-LPETG protein.
2.4 Preparation of LgBiT-long-LPET-4G-SmBiT protein
And (3) replacing the recombinant expression vector pET/LgBiT in the step (2.1) with LgBiT-long-LPET-4G-SmBiT, and obtaining a transformed strain BL21 (DE 3) -pET/LgBiT-long-LPET-4G-SmBiT without changing other steps, thereby further obtaining LgBiT-long-LPET-4G-SmBiT protein.
3. Luciferase activity assay of various proteins:
3.1 determination of background luciferase Activity of proteins
And (3) carrying out gradient dilution on LgBiT, lgBiT-long-LPETG and LgBiT-long-LPET-4G-SmBiT proteins in the second step, wherein each protein is diluted to 10nmol/l, 1.0nmol/l and 0.1nmol/l respectively, and 0.1% Bovine Serum Albumin (BSA) and 0.01% TWEEN-20 are added into Phosphate Buffered Saline (PBS) as a diluent.
The diluted samples were individually taken in 384-well plates (10. Mu.l/well), nanoLuc substrate stock solution purchased from Promega was diluted 100-fold with the above-mentioned dilution, and then added to 384-well plates (10. Mu.l/well), and after mixing, bioluminescence was measured on an enzyme-labeled instrument immediately. The background luciferase activity of LgBiT-LPETG was very high (up to about 30% of the HiBiT post complementation activity), indicating that it was unsuitable for use as a substrate for Sortase a. The background luciferase activity of LgBiT-long-LPETG is very low and is similar to that of the original LgBiT (figure 2B), which shows that LgBiT-long-LPETG can be used as a substrate of Sortase A. The ligation product LgBiT-long-LPET-4G-SmBiT itself was expected to have high luciferase activity (FIG. 2A), and the specific activity was measured to be 4.1X10 5 RLU/fmol is only slightly lower than HiBiT complementary LgBiT-long-LPETG. Thus, 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.
3.2 determination of protein luciferase Activity after HiBiT complementation
LgBiT and LgBiT-long-LPETG proteins in the second step were diluted to 20nmol/l, 2.0nmol/l and 0.2nmol/l respectively with the above-mentioned dilutions, and then these diluted samples were mixed with an equal volume of 80nmol/l HiBiT (formulated with the above-mentioned dilutions), left at room temperature for 10min, added to 384 well plates (10. Mu.l/well), and diluted nanoLuc substrate (10. Mu.l/well) was added, and immediately after mixing, bioluminescence was measured on a microplate reader. As a result, as shown in FIG. 2B, the luciferase activity of LgBiT-long-LPETG was recovered after complementation with HiBiT (FIG. 2B), and the specific activity was measured to be 5.5X10 5 RLU/fmol is almost the same as luciferase activity after the original LgBiT is complementary to HiBiT.
4. Recombinant expression, separation and purification of circular Sortase a:
sortase a is derived from staphylococcus aureus (staphylococcus aureus) and recombinantly expresses the active part consisting of amino acids 60-206. Amino acid residues 60-206 of Sortase A were fused to split introns (split intein) which catalyze the formation of peptide bonds at the N-and C-termini of linear Sortase A, thereby obtaining a more stable circular Sortase A, according to methods previously reported in the present laboratory (see references 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 3and 4using NanoLuc complementation.Amino Acids,50:1111-1119). After induction of expression in E.coli, purification was performed by DEAE anion exchange chromatography and SP cation exchange chromatography, the resulting circular Sortase A was identified as a single band by SDS-PAGE, quantified by UV absorption at 280nm (extinction coefficient 13490 l/mol/cm), and then sub-packed (20-50. Mu.l/tube) and frozen at-80℃for subsequent experiments to obtain recombinant circular Sortase A.
5. Recombinant circular Sortase a catalyzes LgBiT-long-LPETG and 4G-SmBiT ligation by bioluminescence assay:
the preheated LgBiT-long-LPETG, 4G-SmBiT and recombinant circular Sortase A are combined in a connection buffer (100 mmol/l Tris-Cl, pH7.5,10mmol/l CaCl) 2 0.1% BSA) and the mixing ratio is shown in Table 2, and the ligation reaction is performed at 25 ℃.
TABLE 2 mixing ratio of LgBiT-long-LPETG, 4G-SmBiT and recombinant circular Sortase A
LgBiT-long-LPETG(μmol/l) 4G-SmBiT(μmol/l) SortaseA(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
At various reaction times (0 min, 5 min, 10 min, 15 min, 20 min, 25 min) 10. Mu.l of the reaction mixture was taken out, 100-fold diluted in 990. Mu.l of diluent (PBS in 0.1% BSA and 0.01% TWEEN-20) and 10. Mu.l of diluted reaction mixture was taken out therefrom and added to 990. Mu.l of diluent for 100-fold dilution to give 10000-fold diluted reaction mixture. 10000-fold diluted reaction mixtures were added to 384-well plates (10. Mu.l/well), and nanoLuc substrates (10. Mu.l/well) 100-fold diluted with the dilution were added thereto, and immediately after mixing, bioluminescence was measured on an enzyme-labeled instrument. As shown in FIG. 2C, when LgBiT-long-LPETG, 4G-SmBiT and recombinant circular Sortase A were all present, the measured bioluminescence increased linearly with reaction time and the rate of luminescence increase was proportional to the concentration of Sortase A used. However, no change in luminescence was observed in any of the three (fig. 2C). The recombinant circular Sortase A can catalyze the connection of LgBiT-long-LPETG and 4G-SmBiT, so that a connection product with luciferase activity is generated.
Further, a high concentration substrate was used for a long time ligation reaction, samples were taken at different reaction times, 100000-fold diluted, and bioluminescence was measured, and the concentration of ligation product in the reaction mixture was calculated based on the ratio of ligation product. As shown in FIG. 2D, the concentration of ligation product reached a maximum at 6h, about 40. Mu. Mol/l, i.e., about 80% of LgBiT-long-LPETG was ligated with 4G-SmBiT. As a result of SDS-PAGE examination of the ligation reaction mixture, as shown in FIG. 2E, the LgBiT-long-LPETG band (shown by asterisks) was found to gradually decrease in brightness with time, while a slightly larger band (shown by the well, which is a ligation product band and a circular Sortase A band, which overlap) was found to gradually increase in brightness with time (FIG. 2E), indicating that Sortase A was indeed able to catalyze the ligation of LgBiT-long-LPETG with 4G-SmBiT.
Example 2 verification of novel methods for protein ligase Activity based on bioluminescence Using plant Butelase-1
1. Design of ligation type LgBiT and SmBiT for determining Butelase-1 ligase Activity:
according to the recognition sequence of Butelase-1, which is NHV three amino acids, designing a connection type LgBiT for Butelase-1, introducing the recognition sequence into the C-end of the LgBiT, adding a connection peptide consisting of 3 Glys (figure 3A), and naming the recognition sequence as LgBiT-NHV, wherein the amino acid sequence is shown as a Seq ID NO.8 in a sequence table, and the coding nucleotide sequence is shown as a Seq ID NO.7 in the sequence table. According to the nucleophilic attack sequence of Butelase-1 as GI, the two amino acids are introduced into the N-end of SmBiT, and 2 Gly are added as a connector, which is named GI-SmBiT (figure 3A), and the amino acid sequence is shown as the sequence table of Seq ID No. 16. The expected connection product of LgBiT-NHV and GI-SmBiT is named LgBiT-NGI-SmBiT (figure 3A), the amino acid sequence of the connection product is shown as SEQ ID NO.14 in a sequence table, and the coding nucleotide sequence of the connection product is shown as SEQ ID NO.13 in the sequence table.
2. Preparation and purification of proteins
Chemical synthesis and isolation and purification of GI-SmBiT were carried out as described in example 1. Expression vector construction, recombinant expression and separation and purification of LgBiT-NHV and LgBiT-NGI-SmBiT were performed as described in example 1. After purification, lgBiT-NHV and LgBiT-NGI-SmBiT were single bands on SDS-PAGE, and the sizes were as expected (FIG. 3B).
3. Luciferase activity assay of proteins:
the results of the luciferase activity assays of LgBiT-NHV and LgBiT-NGI-SmBiT performed in accordance with the method of example 1 are shown in FIG. 3B, and the results show that the recombinant LgBiT-NGI-SmBiT itself has very high luciferase activity (FIG. 3B), and the specific activity of the assay is 4.9X10 5 RLU/fmol. However, lgBiT-NHV itself has very low luciferase activity and can recover the luciferase activity only after complementation with HiBiT (FIG. 3B), and the specific activity is measured to be 5.6X10 5 RLU/fmol. LgBiT-NHV and GI-SmBiT are therefore suitable as ligation substrates for Butelase-1.
4. Preparing a butterfly bean crude extract and measuring the activity of ligase:
preparation of crude extract of butterfly beans
Butelase-1 is derived from the tropical plant pteridopsis variabilis (C.ternatea). 1g of the leaves and twigs of the pteridopsis variabilis are weighed, sheared, added with 4ml of extracting solution, fully ground in a mortar, centrifuged (10000 g,5 min) and the supernatant is taken, namely the crude extracting solution of the pteridopsis variabilis. A crude extract of pteridopsis japonica was prepared for ligase activity assay using PBS and a reducing reaction solution (20 mmol/l phosphate, pH6.5,1.0mmol/l EDTA,5.0mmol/l beta-mercaptoethanol) as the extracts, respectively.
The preheated LgBiT-NHV, GI-SmBiT and the crude sphenoid extract were mixed with the respective reduction reaction solutions (mixing ratio is shown in Table 3), ligation was performed at 25℃and 10. Mu.l of the reaction mixture was taken out for 3000-fold dilution at different reaction times (0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min), and the bioluminescence was measured, and the results were shown in FIG. 3C. The group of reactions with LgBiT-NHV, GI-SmBiT and crude spherulitic extract all had a gradual increase in bioluminescence measured over time, whether in PBS or in the reducing reaction (FIG. 3C). However, no increase in luminescence was observed in the control group lacking GI-SmBiT or the crude extract of pterocarpus (FIG. 3C). Therefore, butelase-1 in the crude extract of pteroceltis is presumed to catalyze the ligation of LgBiT-NHV and GI-SmBiT, thereby producing a ligation product with luciferase activity. However, the late bioluminescence gradually decreased with a high proportion of crude spheroplasts in the reaction group (FIG. 3C), with a maximum conversion of LgBiT-NHV of only about 2%, which may be caused by hydrolysis of the ligation products by some proteases in the crude extract.
TABLE 3 mixing ratio of LgBiT-NHV, GI-SmBiT and crude spherule extract
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
5. Preparation of partially purified Butelase-1 and ligase Activity determination:
butelase-1 was purified from the crude extract of Pipteroma japonicum according to the method previously reported (Nguyen GK, wang S, qia Y, hemu X, lian Y, tam JP. (2014) Butelase 1is an Asx-specific ligase enabling peptide macrocyclization and synhesis. Nat Chem Biol 10:732-738.Hemu X,Zhang X,Bi X,Liu CF,Tam JP. (2019) Butelase1-Mediated Ligation ofPeptides and proteins. Methods Mol Biol 2012:83-109), and the content of Butelase-1 was about 20% by SDS-PAGE identification of the partially purified Butelase-1 after purification by cationic chromatography. The pre-warmed LgBiT-NHV, GI-SmBiT and partially purified Butelase-1 were mixed in PBS containing 0.1% bsa and ligation was performed at 25 ℃. 10 μl of the reaction mixture was withdrawn at various reaction times for 10000-fold dilution, and 10 μl of the assay bioluminescence was taken. As shown in FIG. 3D, the concentration of the reaction product increased substantially linearly over 25min, especially for the group with low Butelase-1 concentration. In the highest Butelase-1 concentration group, the ligation product concentration reached about 0.6. Mu. Mol/l at 25min, i.e.the conversion of LgBiT-NHV was about 12%.
Further long-term ligation with high concentrations of substrate, bioluminescence assay found that the concentration of 5h ligation product had reached about 14. Mu. Mol/l (FIG. 3E), i.e., the conversion of LgBiT-NHV reached about 60%. SDS-PAGE showed that the intensity of the LgBiT-NHV band gradually decreased during the reaction, while the intensity of a slightly larger band (indicated by asterisks, the ligation product) gradually increased (FIG. 3E), indicating that Butelase-1 did catalyze the ligation of LgBiT-NHV and GI-SmBiT.
Example 3 screening of novel AEP-type protein ligases Using bioluminescence-based assay for protein ligase Activity
Butelase-1 belongs to the class of asparagine proteases (asparaginyl endopeptidase, abbreviated AEP) protein ligases. In order to screen out new AEP type protein ligase for application development, the invention establishes an efficient method for screening AEP type protein ligase from plant crude extract or other sources.
In this example, leaves or flowers of 80 common plants were collected by themselves, 1g of plant tissue was weighed, and 2ml of PBS was added for grinding, and the supernatant was centrifuged to obtain a crude extract. The crude extracts of these 80 plants were screened in the proportions shown in Table 4 using LgBiT-NHV and GI-SmBiT as substrates, and the results are shown in Table 5. After the first round of screening, the potential positive samples (bioluminescence increased more than 2-fold over 0 min after 5 min or 10 min of reaction) were again validated. As shown in fig. 4, the present example found significant AEP-type protein ligase activity in the crude extract of 5 plants, including allium fistulosum (Zephyranthes candida), hibiscus (Hibiscus syriacus), sparrow (Angelonia angustifolia), hosta plantaginea (Hosta plantaginea) and winter jasmine (Jasminum nudiflorum). However, these protein ligases may only be expressed in specific tissues of these plants, for example, very high AEP-type protein ligases activity was detected in flowers and stems of Allium fistulosum, but very low activity in leaves and bulbs; the hibiscus flower has very high AEP type protein ligase activity, but also has very low activity in leaves, which indicates that the protein ligase in some plants is only expressed in specific parts, and the protein ligase can be identified and purified from the tissues only.
TABLE 4 mixing ratio of LgBiT-NHV, GI-SmBiT and crude extracts of plants
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
TABLE 5 detection results of different detection sites of different plants
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Example 4 screening of PEP-type protein ligase Using a method for measuring protein ligase Activity based on bioluminescence
In addition to the AEP type protein ligase, 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. As shown in FIG. 5A, some of the cyclic peptides mature peptides are Pro residues at the C-terminus, presumably by the catalytic synthesis of the Pro-type protein ligase of the prolyl protease (PEP) type.
In order to detect whether a PEP type protein ligase exists in a plant, as shown in FIG. 5B, the PIQ sequence is introduced at the C-end of LgBiT through a connecting peptide consisting of three Glys, and is named LgBiT-PIQ as a possible recognition site of the PEP type protein ligase, the amino acid sequence of the protein ligase is shown as SEQ ID NO.10 in a sequence table, and the coding sequence of the protein ligase is shown as SEQ ID NO.9 in the sequence table. A hydrophobic Phe and three Gly residues are introduced into the N-end of SmBiT and are used as nucleophilic attack sequences possibly recognized by PEP type protein ligase, the sequence is named FG-SmBiT, and the amino acid sequence of the sequence is shown as Seq ID No.17 in a sequence table.
LgBiT-PIQ and FG-SmBiT were prepared, respectively, according to the method of example 1, and the background luciferase activity of LgBiT-PIQ and the luciferase activity after complementation with HiBiT were determined. The results show that LgBiT-PIQ has low background luciferase activity, but can recover the luciferase activity after being complemented with HiBiT, and the specific activity is measured to be 4.3X10 5 RLU/fmol。
The 80 crude extracts of the plants of example 3 were screened with different ratios (see Table 6) of LgBiT-PIQ and FG-SmBiT (FIGS. 5C-F) and found to have significant PEP-type protein ligase activity in 4 plants, including Peacock (Angelonia angustifolia), flower She Qingmu (Aucuba japonica var variegate), herba Eupatorii (Eupatorium fortunei) and Duck grass (Linderniaantipoda). Thus, various types of protein ligases can be rapidly screened and identified from plants or other sources using methods based on bioluminescence assay of protein ligase activity.
TABLE 6 mixing ratio of LgBiT-PIQ, FG-SmBiT and crude plant extract
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
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Sequence listing
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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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (Artificial Sequence)
<400> 17
Phe Gly Gly Gly Val Thr Gly Tyr Arg Leu Phe Glu Glu Ile Leu
1 5 10 15

Claims (8)

1. A composition for determining protein linked enzyme activity comprising a corresponding linked LgBiT protein and linked SmBiT; the connection type LgBiT protein is characterized in that an amino acid sequence recognized by a protein ligase to be detected is fused at the C-terminal of the LgBiT; the connecting SmBiT is characterized in that a nucleophilic attack sequence recognized by a protein ligase to be detected is introduced into the N-end of the SmBiT.
2. The composition of claim 1, wherein: when the protein ligase to be detected is Sortase A ligase, the LgBiT connecting protein is LgBiT-long-LPETG, and the amino acid sequence of the LgBiT connecting protein is shown as Seq ID No. 6; the SmBiT connection protein is 4G-SmBiT, and the amino acid sequence of the SmBiT connection protein is shown as Seq ID No. 15.
3. The composition of claim 1, wherein when the protein ligase to be tested is an asparagine protease type protein ligase, the LgBiT ligase is LgBiT-NHV having an amino acid sequence of Seq ID No. 8; the amino acid sequence of the SmBiT connection protein GI-SmBiT is shown as Seq ID No. 16.
4. The method according to claim 1, wherein: when the protein ligase to be detected is prolyl protease type protein ligase, the LgBiT connecting protein is LgBiT-PIQ, and the amino acid sequence of the LgBiT connecting protein is shown as Seq ID No. 10; the SmBiT connection protein is FG-SmBiT, and the amino acid sequence of the SmBiT connection protein is shown as Seq ID No. 17.
5. A method for determining the activity of various protein ligases based on bioluminescence, characterized in that a composition according to any one of claims 1 to 5 is used.
6. The method according to claim 5, comprising the step of mixing the diluted solution of the composition according to any one of claims 1 to 5 with a protein ligase to be tested to react, and performing a bioluminescence assay on the reaction solution.
7. A composition according to claims 1-4 or a method according to any of claims 5-6 for use in any of the following:
1) The application in preparing the product for measuring the activity of the protein ligase;
2) The application in the determination of protein ligase activity;
3) Use in screening protein ligases of plant, animal or microbial origin;
4) Use in the identification of protein ligases of plant, animal or microbial origin;
5) Use in the characterization of a protein ligase;
6) Application in the activity tracking of protein ligase;
7) Use in a protein ligase inhibitor screen.
8. The use according to claim 7, wherein the protein ligase is AEP-type protein ligase, PEP-type protein ligase or Sortase a ligase.
CN202210120982.2A 2021-10-28 2022-02-09 Composition, method and application for measuring protein ligase activity based on bioluminescence Pending CN116042766A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111117977A (en) * 2020-01-06 2020-05-08 华南理工大学 Recombinant polypeptide linked zymogen, preparation method, activation method and application thereof
CN112352055A (en) * 2018-02-26 2021-02-09 艾恩德霍芬技术大学 Bioluminescent biosensors for detecting and quantifying biomolecules or ligands in solution

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112352055A (en) * 2018-02-26 2021-02-09 艾恩德霍芬技术大学 Bioluminescent biosensors for detecting and quantifying biomolecules or ligands in solution
CN111117977A (en) * 2020-01-06 2020-05-08 华南理工大学 Recombinant polypeptide linked zymogen, preparation method, activation method and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JIA-HUI WANG等: "Rapid preparation of bioluminescent tracers for relaxin family peptides using sortase-catalysed ligation", AMINO ACIDS, vol. 49, 19 June 2017 (2017-06-19), XP036299802, DOI: 10.1007/s00726-017-2455-9 *
NING LI等: "An enzyme-mediated protein-fragment complementation assay for substrate screening of sortase A", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, vol. 486, no. 2, 29 April 2017 (2017-04-29) *
T M SIMON TANG等: "Asparaginyl endopeptidases: enzymology, applications and limitations", ORG BIOMOL CHEM, vol. 19, no. 23, 16 June 2021 (2021-06-16) *

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