Protein ligase and application thereof in catalysis of polypeptide connection
By recombinantly expressing and purifying the ZcAEP1 enzyme from Onion in Escherichia coli, the problem that higher plant-derived AEP-type protein ligase is difficult to express efficiently in Escherichia coli, and high-viability protein ligase under low temperature conditions is achieved. Application of protein labeling and polypeptide ligation.
Patent Information
- Application Number
- CN202310753711.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-21
- Publication Date
- 2024-12-24
AI Technical Summary
The asparagine endonuclease (AEP type protein ligase) from the prior art is difficult to express efficiently recombinantly in E. coli, which limits the development and utilization of such protein ligases, and the extraction process takes time and yield. Low, no commercial protein ligase appears.
A variety of plant crude extracts were screened through NanoLuc luciferase activity complementation technology, ZcAEP1 enzyme from Onion Lotus was identified, and recombinantly expressed and purified in E. coli, and its self-activation method was established under low temperature conditions, and its confirmation was confirmed. It has high protein ligase activity.
AEP-type protein ligase derived from AEP in Escherichia coli is achieved, ensuring that it has broad prospects for protein labeling and peptide ligation under low temperature conditions.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technology and method of genetic engineering and enzymatic characterization, and relates to a protein ligase and its application in catalyzing polypeptide connection. Specifically, the present invention relates to recombinant expression of an allium sibiricum-derived asparagine protein endonuclease in Escherichia coli, and the enzyme has protein ligase activity. Background Art
[0002] Protein ligase (or polypeptide ligase) is a special type of protein endonuclease, which can connect two peptide chains together through peptide bonds through a transpeptidation mechanism, or form a peptide chain into a cyclic peptide through a peptide bond. Among them, asparaginyl endopeptidase (AEP) type protein ligases from higher plants, such as Butelase-1 from Clitoria ternatea and OaAEP1 from Oldenlandia affinis, have attracted much attention due to their high catalytic activity. They have been widely used in the connection, labeling and cyclization of proteins and polypeptides, and have important application development value. However, plant extraction is easily limited by raw materials, the extraction process is time-consuming and the yield is low. Studies have shown that it takes 3 days to obtain only 5 mg of natural Butelase-1 from 1 kg of pods. At present, only a few AEP-type protein ligases have been identified from higher plants, and these identified protein ligases are difficult to efficiently recombinantly express in Escherichia coli, which limits the development and utilization of this type of protein ligase, resulting in the lack of commercial sales of this type of protein ligase. At present, there is an urgent need for an AEP protein ligase that can be expressed in large quantities in Escherichia coli and can effectively catalyze the connection of polypeptides. Summary of the invention
[0003] In order to solve the above technical problems, the present invention provides a protein ligase and its application in catalyzing polypeptide ligation. The inventors have developed a new method for determining the activity of protein ligases based on NanoLuc luciferase activity complementation technology (NanoBiT). Through screening of AEP-type protein ligase activity in crude extracts of various plants, and combined with transcriptome sequencing, a protein homologous to other plant AEPs was identified from the monocot Zephyranthes candida, named ZcAEP1, and it is speculated that it may be a protein ligase. In order to confirm whether it is a protein ligase or a hydrolase, it can only be verified by obtaining a purified recombinant enzyme through heterologous recombinant expression.
[0004] The present invention establishes a method for recombinantly expressing ZcAEP1 proenzyme using Escherichia coli, as well as its purification and in vitro self-activation. Protein ligase activity assay confirms that the proenzyme has high protein ligase activity and can function under low temperature conditions (such as 0°C). It has broad application prospects in various protein labeling, especially protein ligation.
[0005] In a first aspect, the present invention provides a protein ligase comprising an amino acid sequence as shown in SEQ ID NO:1.
[0006] In a preferred embodiment, the protein ligase has an amino acid sequence as shown in SEQ ID NO:1.
[0007] The protein ligase of the present invention is also called polypeptide ligase.
[0008] In a preferred embodiment, the protein ligase of the present invention is substantially a zymogen.
[0009] The second aspect of the present invention provides a nucleotide sequence encoding the protein ligase as described in the first aspect of the present invention.
[0010] Preferably, the nucleotide sequence comprises or has the nucleotide sequence shown in SEQ ID NO:7.
[0011] The third aspect of the present invention provides a recombinant expression vector comprising a nucleotide sequence encoding the protein ligase as described in the first aspect of the present invention or the nucleotide sequence as described in the second aspect of the present invention.
[0012] In a preferred embodiment, the recombinant expression vector is pET28a.
[0013] In a preferred embodiment, the recombinant expression vector is fused with a sequence encoding a 6×His tag.
[0014] The fourth aspect of the present invention provides a transformant, which comprises the recombinant expression vector as described in the third aspect of the present invention.
[0015] In a preferred embodiment, the transformant is a prokaryotic organism.
[0016] In a preferred embodiment, the transformant is Escherichia coli, such as Escherichia coli Shuffle-T7 strain.
[0017] The fifth aspect of the present invention provides a method for preparing a protein ligase, which comprises extracting the protein ligase from the transformant as described in the fourth aspect of the present invention under conditions suitable for obtaining the protein ligase.
[0018] In a preferred embodiment, the fermented transformant is resuspended in an appropriate amount of lysis buffer, and then the transformant is lysed by ultrasonication, centrifuged and purified.
[0019] In a preferred embodiment, the lysis buffer comprises 50 mM Tris-Cl, pH 8.0, 100 mM NaCl, 0.1% Triton X-100).
[0020] In a preferred embodiment, the centrifugation is 12000 g for 30 min.
[0021] In a preferred embodiment, the purification is carried out by Ni 2+ Column purification.
[0022] The sixth aspect of the present invention provides a method for activating a protein ligase, comprising mixing the protein ligase as described in the first aspect of the present invention with a sodium acetate buffer having a pH of 4.0 to 4.8, and incubating the mixture at 15 to 37° C. to obtain an activated protein ligase.
[0023] In a preferred embodiment, the volume ratio of the protein ligase to the sodium acetate buffer is 1:1.
[0024] In a preferred embodiment, the concentration of the sodium acetate buffer is 100-300 mM, such as 150 mM, 200 mM or 250 mM.
[0025] In a preferred embodiment, the pH of the sodium acetate buffer is 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7 or 4.8.
[0026] In a preferred embodiment, the incubation is carried out at 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C or 36°C.
[0027] The seventh aspect of the present invention provides an activated protein ligase, which is obtained by the activation method as described in the sixth aspect of the present invention.
[0028] The eighth aspect of the present invention provides a method for catalyzing polypeptide ligation, which comprises ligating polypeptides using the protein ligase described in the first aspect of the present invention or the activated protein ligase described in the seventh aspect of the present invention.
[0029] In a preferred embodiment, the polypeptides are linked at pH 6.5-8.5, for example, pH 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4 or 8.5.
[0030] In a preferred embodiment, the connection time is 5 min to 2.5 h, for example 10 min, 20 min, 30 min, 40 min, 50 min, 1 h, 1.5 h, 2 h or 2.5 h.
[0031] In a preferred embodiment, the polypeptides are linked at a temperature between 0°C and 25°C; for example, at 5°C, 8°C, 10°C, 12°C, 15°C, 17°C, 18°C, 20°C or 22°C.
[0032] In a preferred embodiment, the ligation is performed in a buffer of phosphate buffer or phosphate buffer.
[0033] In a preferred embodiment, the concentration ratio of the protein ligase or the activated protein ligase to the polypeptide is 1:(1-10)×10 3 For example, the concentration ratio of the protein ligase or the activated protein ligase to the polypeptide is 1:2×10 3 , 1:4×10 3 or 1:8×10 3 .
[0034] In a preferred embodiment, the concentration of the protein ligase or the activated protein ligase is 20-100 nM, such as 40 nM, 50 nM or 80 nM.
[0035] The polypeptide comprises a recognition sequence polypeptide and / or a nucleophilic attack polypeptide; for example, the polypeptide is a recognition sequence polypeptide and an affinity attack polypeptide, and the method catalyzes the connection between the recognition sequence polypeptide and the affinity attack polypeptide.
[0036] In a preferred embodiment, the recognition sequence polypeptide is linked to the N-terminus of the nucleophilic attack polypeptide.
[0037] In a preferred embodiment, the third amino acid at the C-terminus of the recognition sequence polypeptide is asparagine.
[0038] In a preferred embodiment, the recognition sequence polypeptide is an amino acid sequence as shown in any one of SEQ ID NOs: 2 to 5.
[0039] In a preferred embodiment, the nucleophilic attack polypeptide is an amino acid sequence as shown in SEQ ID NO:6.
[0040] In a preferred embodiment, the polypeptide is a luciferase such as LgBiT-NHV and / or GI-SmBiT, wherein LgBiT-NHV and GI-SmBiT are as defined in CN116042766A.
[0041] In a preferred embodiment, when the polypeptide includes a recognition sequence polypeptide and / or a nucleophilic attack polypeptide, the concentration of the protein ligase or the activated protein ligase is 100 nM, the concentration of the recognition sequence polypeptide is 100 μM, and the concentration of the nucleophilic attack polypeptide is 1000 μM.
[0042] In a preferred embodiment, when the polypeptide is a luciferase such as LgBiT-NHV and / or GI-SmBiT, the concentration of the protein ligase or the activated protein ligase is 25 nM, the concentration of the LgBiT-NHV is 50 μM, and the concentration of the GI-SmBiT is 200 μM.
[0043] The ninth aspect of the present invention provides a protein ligase as described in the first aspect of the present invention, the nucleotide sequence as described in the second aspect of the present invention, the recombinant expression vector as described in the third aspect of the present invention, the transformant as described in the fourth aspect of the present invention, or the activated protein ligase as described in the seventh aspect of the present invention for use in catalyzing polypeptide ligation, polypeptide cyclization or polypeptide labeling.
[0044] In a preferred embodiment, the polypeptide is as defined in the method according to the eighth aspect of the present invention.
[0045] The present invention aims to: 1) provide an AEP-type protein ligase derived from Allium truncatum expressed recombinantly in Escherichia coli (named "recombinant ZcAEP1") and a preparation method thereof; 2) provide multiple uses of the recombinant AEP-type protein ligase, including catalyzing the labeling, connection and cyclization of various proteins and polypeptides.
[0046] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.
[0047] The reagents and raw materials used in the present invention are commercially available.
[0048] The positive and progressive effects of the present invention are: the present invention establishes a method for recombinantly expressing ZcAEP1 proenzyme using Escherichia coli, and its purification and in vitro self-activation, and the protein ligase of the present invention can be efficiently expressed in recombinant Escherichia coli. The protein ligase activity assay confirms that it has high protein ligase activity and can function under low temperature conditions (such as 0°C), and has broad application prospects in various protein labeling, especially protein ligation. BRIEF DESCRIPTION OF THE DRAWINGS
[0049] Figure 1 The nucleotide and amino acid sequences encoding 6×His-ZcAEP1 are shown in FIG.
[0050] Figure 2 SDS-PAGE analysis of the recombinant expression and separation and purification of ZcAEP1 proenzyme, where lane b, pre-induction bacteria; lane a, post-induction bacteria; lane s, supernatant after ultrasonic cell disruption; lane p, precipitate after ultrasonic cell disruption; lane f, flow-through; lane w1, equilibrium solution elution fraction; lane w2, 20 mM imidazole elution fraction; lane e, 100 mM imidazole elution fraction. The 6×His-ZcAEP1 band is indicated by an asterisk.
[0051] Figure 3 The self-activation of recombinant ZcAEP1 proenzyme was monitored by SDS-PAGE, wherein the recombinant ZcAEP1 proenzyme was mixed with an equal volume of 200 mM sodium acetate buffer (pH 4.4) and incubated at 25°C. At the designated time, 10 μl of the self-activation mixture was mixed with 5 μl of loading buffer and boiled before SDS-PAGE analysis. The separation gel concentration was 15%, and after electrophoresis, it was stained with Coomassie Brilliant Blue R250.
[0052] FIG4 shows the connection of LgBiT-NHV and GI-SmBiT catalyzed by self-activation of recombinant ZcAEP1 proenzyme monitored by bioluminescence and SDS-PAGE, wherein the recombinant ZcAEP1 proenzyme was mixed with an equal volume of 200 mM sodium acetate buffer (pH 4.4) and incubated at 25° C. At the designated time, 10 μl of the self-activation mixture was taken and diluted 50 times with enzyme dilution buffer (200 mM PB, pH 7.0, 0.2% BSA) for protein ligase activity assay. 10 μl of the diluted enzyme was mixed with an equal volume of substrate (100 μM LgBiT-NHV and 400 μM GI-SmBiT), the connection reaction was started at 15° C. and 10 μl of the reaction mixture was taken out at 5 min or 60 min, and the bioluminescence assay was performed after dilution 100,000 times ( Figure 4A ); or after 60 minutes of connection, take out 10 μl of the reaction mixture, mix it with 40 μl of loading buffer, boil it, and then take out 10 μl of the sample for SDS-PAGE analysis ( Figure 4B ). The concentration of the separation gel was 15%, and after electrophoresis, it was stained with Coomassie Brilliant Blue R250.
[0053] Figure 5 shows the effect of pH and temperature on the activity of recombinant ZcAEP1 ligase, where Figure 5A: Effect of reaction pH on the initial ligation velocity of recombinant ZcAEP1. Recombinant ZcAEP1 (2 nM) catalyzed the ligation reaction of LgBiT-NHV (5 μM) and GI-SmBiT (10 μM) at 20°C in ligation buffer (200 mM PB containing 0.1% BSA) at different pH values. After 10 min of reaction, 10 μl of the reaction mixture was taken out and diluted 2000 times with NanoBiT dilution buffer to measure bioluminescence. The measured data are expressed as mean ± standard deviation (n = 3). Figure 5B and Figure 5C :Through bioluminescence ( Figure 5B ) and SDS-PAGE analysis ( Figure 5C ) The effect of pH on the accumulation of ligation products by recombinant ZcAEP1 was analyzed. Recombinant ZcAEP1 (15 nM) catalyzed the ligation reaction of LgBiT-NHV (25 μM) and GI-SmBiT (100 μM) at 20°C in ligation buffer (200 mM PB) with different pH values. After 2.5 h of reaction, 10 μl of the reaction mixture was taken out and diluted 10,000 times before measuring the bioluminescence ( Figure 5B ), or mixed with 30 μl loading buffer and analyzed by SDS-PAGE ( Figure 5C ). Figure 5D and Figure 5E : Recombinant ZcAEP1 at pH 7.0 ( Figure 5D ) and pH7.5( Figure 5E ) conditions. Recombinant ZcAEP1 was diluted with 200 mM PB containing 0.1% BSA to a final concentration of 6 nM and incubated at different temperatures. At the specified time, 5 μl of enzyme solution was taken out and mixed with an equal volume of substrate (10 μM LgBiT-NHV and 20 μM GI-SmBiT), reacted at room temperature for 10 min, and then diluted 2000 times with NanoBiT dilution buffer and measured for bioluminescence.
[0054] Figure 6 shows the bioluminescence ( Fig. 6A ) and SDS-PAGE( Figure 6B ) monitor the ligase activity of recombinant ZcAEP1 under low temperature conditions. Recombinant ZcAEP1 catalyzes the ligation reaction of GI-SmBiT and LgBiT-NHV at 10°C or 0°C (substrate and enzyme concentrations are marked in the figure). At the specified time, 10 μl of the ligation mixture was taken out and diluted 100,000 times with NanoBiT dilution buffer and then the bioluminescence was measured ( Fig. 6A ), or mixed with 40 μl of SDS gel loading buffer for SDS-PAGE analysis ( Figure 6B ).
[0055] FIG. 7 shows the effect of recombinant ZcAEP1 on the substrate LgBiT-NHV ( Fig. 7A) and GI-SmBiT( Figure 7B ) was used to measure the enzymatic parameters of the reaction. Recombinant ZcAEP1 catalyzed the ligation reaction of GI-SmBiT and LgBiT-NHV in a reaction buffer containing 0.1% BSA (substrate and enzyme concentrations are marked in the figure). After reacting at 20°C for 10 minutes, 10 μl of the reaction mixture was taken out and diluted 10,000 times with NanoBiT dilution buffer to measure the bioluminescence. The initial velocity of the ligation product generated within 10 minutes was calculated based on the bioluminescence and specific activity of the ligation product, expressed as the mean ± standard deviation (n = 3), and converted to V = V using SigmaPlot 10.0 software. max [S] / (K m +[S]) equation fitting.
[0056] FIG8 shows the bimolecular ligation of synthetic peptides catalyzed by ZcAEP1, wherein: Fig. 8A : HPLC analysis of ZcAEP1 catalyzing the bimolecular ligation of GIGGIR with four substrates (YGGGGNHV, YGGGVNHV, YGGVGNHV, YGVGGNHV). 100 nM ZcAEP1 catalyzed the ligation of 100 μM polypeptides as shown in SEQ ID NO: 2-5 and 1000 μM GIGGIR in 200 mM PB (pH 7.5) at 0°C for 20 min. Then 500 μl of the reaction mixture was taken, 13 μl of 50% TFA was added to terminate the reaction, and analyzed by HPLC using a C18 reverse phase column. Figure 8B : HPLC analysis of the effect of reaction time on the ligation of GIGGIR and YGGGGNHV catalyzed by ZcAEP1. 100 nM ZcAEP1 catalyzed the ligation of 100 μM YGGGGNHV and 1000 μM GIGGIR in 200 mM PB (pH 7.5) at 0°C. 500 μl of the reaction mixture was taken at different times, 13 μl of 50% TFA was added to terminate the reaction, and analyzed by HPLC using a C18 reverse phase column. DETAILED DESCRIPTION
[0057] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.
[0058] Example 1: Construction, expression and purification of a strain containing the ZcAEP1 proenzyme gene
[0059] This example aims to illustrate the construction, expression, separation and purification of recombinant ZcAEP1 proenzyme.
[0060] The sequences involved are as follows:
[0061]
[0062] The specific steps of this method are as follows:
[0063] 1.1 Construction of recombinant ZcAEP1 enzyme plasmid
[0064] The amino acid sequence of ZcAEP1 proenzyme is shown in SEQ ID NO: 1. The DNA sequence encoding ZcAEP1 proenzyme (excluding the N-terminal signal peptide) was chemically synthesized at Beijing Qingke Company using Escherichia coli preferred codons. Figure 1 ), as shown in SEQ ID NO: 7. After cutting with restriction endonucleases EcoRI and NdeI, the synthesized DNA fragment was cloned into the E. coli expression vector pET28a (with 6×His tag coding sequence fused upstream) to obtain the expression plasmid pET / 6×His-ZcAEP1, which was confirmed by DNA sequencing.
[0065] 1.2 Construction and expression of recombinant ZcAEP1 proenzyme strain
[0066] The expression vector pET / 6×His-ZcAEP1 was transformed into the E. coli expression strain Shuffle-T7 (Weidi Biotechnology Co., Ltd.), and the transformed E. coli was inoculated into LB liquid medium (containing 100 mg / l ampicillin) and cultured in a shaker at 37°C until the optical density OD600 reached about 0.7. The culture solution was then cooled to 16°C, and the inducer IPTG was added to 0.1 mM, and the culture was continued in a shaker at 16°C for 24 hours.
[0067] 1.3 Isolation and purification of recombinant ZcAEP1 enzyme
[0068] Take the expression culture of the recombinant ZcAEP1 enzyme strain obtained in 1.2, then centrifuge (5000g, 10min) to collect the bacteria, and resuspend the bacteria in an appropriate amount of lysis buffer (50mM Tris-Cl, pH8.0, 100mM NaCl, 0.1% Triton X-100), and then lyse the bacteria by ultrasound. Then centrifuge (12000g, 30min) to take the supernatant, and pass Ni 2+ Column purification. The recombinant enzyme was eluted with 100mM imidazole (dissolved in lysis buffer), and the eluted fractions were collected and dialyzed against dialysis buffer (20mM PB, pH6.5, 100mM NaCl, 5mM β-mercaptoethanol, 1mM EDTA) for 2h. Finally, centrifuge (10000g, 10min) to take the supernatant, add glycerol to a final concentration of 30%, divide (20-50μl / tube) and freeze at -80℃ for subsequent experiments. Samples at each step were analyzed by SDS-PAGE ( Figure 2 ).2+ The eluted zymogen was dialyzed against cold storage solution to remove the imidazole which inhibits the zymogen's self-activation. The recombinant ZcAEP1 zymogen in the storage solution can be stored at -80°C for several months without significant inactivation.
[0069] Example 2: Optimization of ZcAEP1 proenzyme autoactivation conditions
[0070] This example aims to illustrate the specific operation method for optimizing the self-activation conditions of ZcAEP1 proenzyme.
[0071] The specific steps of this method are as follows:
[0072] The purified ZcAEP1 proenzyme (about 0.3 mg / ml) obtained in 1.3 was mixed with equal volumes of 200 mM sodium acetate buffer at different pH values, and self-activated at different temperatures to remove the C-terminal Cap domain. Three self-activation pH values (4.0, 4.4, 4.8) and three self-activation temperatures (15°C, 25°C, 37°C) were set. After incubation for a specified time, 10 μl of the self-activation mixture was taken out, and either mixed with 5 μl of loading buffer for SDS-PAGE analysis, or diluted 50 times with enzyme diluent (200 mM PB, pH 7.0) for ligation activity determination.
[0073] The specific activity assay operation is as follows: the purified zymogen is mixed with an equal volume of activation solution and incubated at a certain temperature for a certain time. The zymogen is mixed with an equal volume of 200 mM sodium acetate buffer (pH 4.4) and incubated at 25°C. SDS-PAGE analysis shows that the 52 kDa zymogen band is rapidly converted to a 40 kDa band within 20 min ( Figure 3 ), but the 40kDa band is relatively stable and can be slowly transformed into a slightly lower band ( Figure 3 ). Therefore, the recombinant ZcAEP1 proenzyme appears to retain the native structure and can be efficiently self-activated under acidic conditions.
[0074] Example 3: Ligase activity assay of ZcAEP1
[0075] This example is intended to illustrate the method of measuring the ligase activity of ZcAEP1 enzyme using luciferase.
[0076] The specific steps of this method are as follows:
[0077] Using LgBiT-NHV and GI-SmBiT as substrates, the inventors used protein ligase activity to monitor the self-activation of ZcAEP1 proenzyme (Figure 4). Among them, LgBiT-NHV and GI-SmBiT are luciferases, and the preparation methods of the two refer to the descriptions in Examples 2 and 3 in CN116042766A. After the proenzyme is self-activated at 25°C for different times, it is sampled and diluted into a neutralization buffer, and then used to catalyze the ligation reaction of LgBiT-NHV and GI-SmBiT. Samples were taken at 5 minutes of the ligation reaction to determine the initial rate of product formation, and samples were taken at 60 minutes to determine the amount of the final ligation product. According to the results of the bioluminescence assay ( Figure 4A ), the ligase activity of the recombinant ZcAEP1 proenzyme increased rapidly after self-activation and reached stability within 30 minutes. In addition, the amount of ligation products generated by the self-activated ZcAEP1 within 5 minutes was as high as 60% of that at 60 minutes ( Figure 4A ), indicating that recombinant ZcAEP1 has high ligase activity. The ligation product was analyzed by SDS-PAGE ( Figure 4B ), after the recombinant proenzyme was self-activated for 10 min or longer, it could convert about 70% of the LgBiT-NHV substrate into a ligation product (marked with an asterisk) within 60 min, indicating that ZcAEP1 is indeed a protein ligase. In summary, the recombinant ZcAEP1 proenzyme can be converted into a highly active protein ligase through self-activation.
[0078] Example 4: Optimal pH and thermal stability of ZcAEP1
[0079] This example aims to illustrate the optimal pH and thermal stability for the ZcAEP1 enzyme to function.
[0080] The specific steps of this method are as follows:
[0081] The inventors used LgBiT-NHV and GI-SmBiT as substrates and tested the performance of recombinant ZcAEP1 by intermolecular ligation at room temperature (15-25°C). First, the effect of pH on its ligase activity was analyzed. FIG. 5A to FIG. 5C ). Recombinant ZcAEP1 showed high ligase activity at pH 6.5-8.5, i.e., the initial velocity of the ligation reaction ( Figure 5A ); however, the accumulation of ligation products during long-term ligation (2.5 h) at high pH was much less ( Figure 5B , Figure 5C ), indicating that the enzyme may be unstable under high pH conditions or have higher hydrolytic activity. Under the optimal condition of pH 7.0, recombinant ZcAEP1 can ultimately convert about 70% of LgBiT-NHV into ligation products ( Figure 5B and Figure 5CThe inventors then determined the effects of different pH and temperature on the stability of ZcAEP1 ( Figure 5D , Figure 5E In pH 7.0 phosphate buffer, the half-life of recombinant ZcAEP1 at 15°C was approximately 100 min ( Figure 5D ). As the temperature increases, its half-life decreases significantly ( Figure 5D In pH 7.5 phosphate buffer, its half-life is only about 40 minutes at 15°C, and the half-life is even shorter when the temperature rises ( Figure 5E ), indicating that the stability of ZcAEP1 decreased under high pH conditions. Comprehensive analysis showed that ZcAEP1 was not stable at room temperature and was easily inactivated, especially under high pH conditions.
[0082] However, the inventors found that the stability of recombinant ZcAEP1 at room temperature was not high enough. Therefore, the inventors used LgBiT-NHV and GI-SmBiT as substrates to test its performance under low temperature conditions. At 10°C or even 0°C, 25nM recombinant ZcAEP1 was used to catalyze the connection of 50μM LgBiT-NHV and 200μM GI-SmBiT. The inventors monitored that the bioluminescence increased rapidly and reached a platform within 1h ( Fig. 6A The ligation rate at 0°C was slightly lower than that at 10°C, but the yields at the two temperatures were comparable: 60-70% of LgBiT-NHV could be converted into ligation products according to bioluminescence assay and SDS-PAGE analysis ( Fig. 6A and Figure 6B ). This shows that ZcAEP1 is a psychrophilic enzyme that can effectively catalyze peptide ligation at low temperatures, even at 0°C (ice bath).
[0083] Example 5: Enzymatic parameters of ZcAEP1-catalyzed ligation reaction of LgBiT-NHV and GI-SmBiT
[0084] This example aims to illustrate how to efficiently exert the activity of ZcAEP1 enzyme ligase.
[0085] The specific steps of this method are as follows:
[0086] The inventors further determined the Km and Vmax values of recombinant ZcAEP1 for the two substrates LgBiT-NHV and GI-SmBiT ( Fig. 7A and Figure 7B ). As the concentration of LgBiT-NHV increases, the initial velocity of the ligation product calculated from the bioluminescence and specific activity of the ligation product shows a typical hyperbolic shape ( Fig. 7A), from which the Km value for this substrate was calculated to be about 54mM and the Vmax value was about 320nM / min. Therefore, in the case of 100μM GI-SmBiT and saturated LgBiT-NHV, one ZcAEP1 can generate about 160 ligation products within 1min. For another substrate GI-SmBiT, the initial rate of product formation catalyzed by recombinant ZcAEP1 also showed a hyperbolic curve ( Figure 7B ), the Km value of the substrate was calculated to be about 96mM, and the Vmax value was about 480nM / min. Therefore, in the case of 100μM LgBiT-NHV and saturated GI-SmBiT, one ZcAEP1 can generate about 240 ligation products within 1min. In summary, recombinant ZcAEP1 can effectively catalyze the intermolecular ligation of LgBiT-NHV and GI-SmBiT, showing a high ligase activity.
[0087] Example 6: Recombinant ZcAEP1 catalyzes bimolecular ligation of synthetic peptides
[0088] This example aims to further illustrate the factors affecting the bimolecular ligation of peptides synthesized by ZcAEP1 enzyme.
[0089] The specific steps of this method are as follows:
[0090] As described in Example 3 above, recombinant ZcAEP1 can catalyze the connection of LgBiT-NHV protein and GI-SmBiT. The inventors further used synthetic peptides to test the effect of substrate sequence on its connection efficiency. Four peptides containing ZcAEP1 recognition sequences (YGVGGNHV, YGGVGNHV, YGGGVNHV and YGGGGNHV, as shown in SEQ ID NOs: 2 to 5, respectively) and nucleophilic attack peptide GIGGIR (as shown in SEQ ID NO: 6) were synthesized by Shanghai Jier Company. The connection of these peptides catalyzed by ZcAEP1 was carried out in 200mM PB (pH 7.5) at 0°C, and the connection system contained 100nM ZcAEP1, 100μM of one of the peptides shown in SEQ ID NOs: 2 to 5, and 1000μM GIGGIR (SEQ ID NO: 6). At designated time (0-40 min), 500 μl of the reaction mixture was taken, 13 μl of 50% TFA was added to terminate the reaction, and then analyzed by RP-HPLC. The ligation yield was analyzed based on the peak area of each component.
[0091] The inventors synthesized a peptide YGGGGNHV with the smallest Gly residues at positions P2-P5 of the substrate, and then introduced a large Val residue at positions P2-P4. Then, recombinant ZcAEP1 was used to catalyze the ligation reaction of these synthetic peptides with another synthetic peptide GIGGIR. Fig. 8A As shown in the figure, under the same ligation conditions, YGGGGNHV and YGVGGNHV (as shown in SEQ ID NO:5) have more ligation products and less hydrolysis products; YGGGVNHV (as shown in SEQ ID NO:3) has the most hydrolysis products; and YGGVGNHV (as shown in SEQ ID NO:4) has the least ligation products. Therefore, the P2 and P3 residues of the substrate have a relatively large influence on the ligation and hydrolysis of ZcAEP1. When the P3 position is a large volume Val, it is not conducive to the ligation reaction, while when the P2 position is Val, the hydrolase activity can be significantly increased. The amino acid residues at the P2 and P3 positions of other AEP-type protein ligases also have an effect on the enzyme activity.
[0092] The inventors then used the optimized substrate YGGGGNHV to study the accumulation of ligation products and hydrolysis products over reaction time ( Figure 8B ). After the ligation reaction started, the ligation product was generated quickly, while the hydrolysis product appeared slowly, indicating that ZcAEP1 was more inclined to the ligation reaction. At 20 minutes, the difference rate of the ligation product reached the highest (about 50%). As the reaction time was further extended, the hydrolysis product increased significantly, but the change of the ligation product was not significant.
Claims
1. A protein ligase having an amino acid sequence as shown in SEQ ID NO:
1.
2. A nucleotide sequence, characterized in that It encodes the protein ligase as claimed in claim 1; preferably, it comprises or has the nucleotide sequence shown in SEQ ID NO:
7.
3. A recombinant expression vector, characterized in that: Containing a nucleotide sequence encoding the protein ligase according to claim 1 or a nucleotide sequence according to claim 2; Preferably, the recombinant expression vector is pET28a, preferably fused with a sequence encoding a 6×His tag.
4. A transformant, characterized in that It comprises the recombinant expression vector as claimed in claim 3; preferably, the transformant is a prokaryotic organism, preferably Escherichia coli, such as Escherichia coli Shuffle-T7 strain.
5. A method for preparing a protein ligase, characterized in that: The protein ligase is extracted from the transformant according to claim 4 under conditions suitable for obtaining the protein ligase.
6. A method for activating a protein ligase, characterized in that: Mixing the protein ligase according to claim 1 with a sodium acetate buffer having a pH of 4.0 to 4.8, and incubating at 15 to 37° C. to obtain an activated protein ligase; Preferably, the volume ratio of the protein ligase to the sodium acetate buffer is 1:1; and / or the concentration of the sodium acetate buffer is 100-300 mM.
7. An activated protein ligase obtained by the activation method according to claim 6.
8. A method for catalyzing polypeptide ligation, characterized in that: The polypeptides are ligated by using the protein ligase according to claim 1 or the activated protein ligase according to claim 7.
9. The method according to claim 8, characterized in that The method satisfies one or more of the following conditions: The peptides were linked at pH 6.5-8.5; The connection time is 5min~2.5h; The peptides were connected at a temperature of 0°C to 25°C; The connection is carried out in a buffer, preferably a phosphate buffer or a phosphate buffer; The concentration ratio of the protein ligase or the activated protein ligase to the polypeptide is 1:(1-10)×10 3 Preferably, the concentration of the protein ligase or the activated protein ligase is 20 to 100 nM.
10. The method according to claim 8 or 9, characterized in that The polypeptide comprises a recognition sequence polypeptide and / or a nucleophilic attack polypeptide; preferably, the third amino acid at the C-terminus of the recognition sequence polypeptide is asparagine; Preferably, the recognition sequence polypeptide is an amino acid sequence as shown in any one of SEQ ID NOs: 2 to 5, and / or the nucleophilic attack polypeptide is an amino acid sequence as shown in SEQ ID NO: 6, or the polypeptide is a luciferase such as LgBiT-NHV and / or GI-SmBiT.
11. Use of the protein ligase according to claim 1, the nucleotide sequence according to claim 2, the recombinant expression vector according to claim 3, the transformant according to claim 4 or the activated protein ligase according to claim 7 in catalyzing polypeptide ligation, polypeptide cyclization or polypeptide labeling.