Sortase A transpeptidase mutant and application thereof
By performing single- or double-point mutations on Sortase A transpeptidase to optimize its amino acid sequence, the efficiency and stability issues of existing Sortase A transpeptidases in industrial and clinical applications have been resolved, resulting in mutants with higher activity and better stability, suitable for large-scale production and industrial use.
Patent Information
- Application Number
- CN202511272871.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-08
- Publication Date
- 2025-11-28
AI Technical Summary
Existing Sortase A transpeptidases suffer from low reaction efficiency, insufficient substrate specificity, and poor tolerance to extreme environments in industrial and clinical applications, which limits their widespread use.
By performing single- or double-point mutations on Sortase A transpeptidase, its amino acid sequence was optimized to obtain mutants with higher activity and thermostability, including K138I, K138V, S116V, and I123P mutation sites. These mutants were then constructed on the pET-29a vector and expressed and purified.
It improves the thermostability and activity of Sortase A transpeptidase, enhances its adaptability to extreme environments, and makes it suitable for large-scale production and industrial use.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of bioengineering technology, and particularly relates to a Sortase A transpeptidase mutant and application thereof. BACKGROUND
[0002] Sortase A transpeptidase isolated from Staphylococcus aureus can specifically recognize and bind the threonine and glycine in the LPXTG sequence at the C-terminal of the protein (X is any amino acid except cysteine and tryptophan), so that the peptide bond is broken, and it is connected to another peptide chain containing a glycine residue at the N-terminal through a peptide bond. Based on this function, Sortase A transpeptidase can be used for protein synthesis and modification.
[0003] In recent years, Sortase A transpeptidase has been widely used in the modification of protein terminals. It is found that any water-soluble modification group of interest can be added to the terminal of the protein by using the transpeptidation reaction catalyzed by Sortase A transpeptidase, including functional groups such as biotin, fluorescein and cross-linking agent. However, wild-type Sortase A transpeptidase has some limitations in practical application, such as low reaction efficiency, insufficient substrate specificity and poor tolerance to some extreme environments (such as high temperature or organic solvents). These problems limit its popularization in industrial production and clinical application. Therefore, it has become one of the current research focuses to optimize the performance of Sortase A transpeptidase by molecular modification and develop mutants with higher activity and better stability.
[0004] Currently, there are studies that can significantly improve the catalytic efficiency and substrate compatibility of Sortase A transpeptidase by site-directed mutagenesis of key amino acid sites. For example, Jeong HJ, Abhiraman GC, Story CM, Ingram JR, Dougan SK (2017) Generation of Ca 2+ -independent sortase A mutants with enhancedactivity for protein and cell surface labeling. PLOS ONE 12 (12): e0189068. discloses 7+SrtA enzyme, which is a truncated body from the 60th to the 206th of the protein sequence of full-length Sortase A transpeptidase (1-206) and contains P94R, D160N, D165A, G167E, K190E, K196T, E105K, E108Q, D124G, Y187L, E189R mutation sites. This mutant realizes Ca 2+In addition to this, combined with directed evolution technology, high-efficiency mutants suitable for different application scenarios can be screened. These improved 7+SrtA enzyme mutants not only meet the needs of basic research, but also may play an important role in the fields of disease diagnosis, drug delivery and synthetic biology, etc.
[0005] However, the existing 7+SrtA enzyme mutants still face many challenges, such as how to further improve their catalytic efficiency, enhance stability and reduce production cost, etc. Therefore, the present application aims to provide a new 7+SrtA enzyme mutant and explore its innovative use in labeling applications, in order to overcome the above shortcomings and promote the development of related technologies. SUMMARY
[0006] The present application aims to solve the existing technical problems and proposes a Sortase A transpeptidase mutant and its application.
[0007] The present application achieves the above-mentioned purposes through the following technical solutions: As a first aspect of the present application, a Sortase A transpeptidase mutant is also included, which is obtained by single mutation or double mutation based on the protein sequence (7+SrtA enzyme) shown in SEQ ID NO. 2, and the protein sequence shown in SEQ ID NO. 2 is a truncation mutant at positions 60-206 of the wild-type Sortase A transpeptidase shown in SEQ ID NO. 1; Taking the sequence of the wild-type Sortase A transpeptidase shown in SEQ ID NO. 1 as a reference, the single mutation site of the Sortase A transpeptidase mutant at least includes one of K138I, K138V, S116V, I123P, F122I, D176P, Q178R, A135K, E85V, D82N, W194Y, K198L, D186N, S116A, E85Q, R151Y, A135N, R99K, V193K, T203K, N127D, T121R, D80A, D80S or T203E; The double mutation site of the Sortase A transpeptidase mutant at least includes one group of (1)-(10): (1) K138V, I123P; (2) K138V, S116V; (3) K138V, F122I; (4) S116V, I123P; (5) S116V, F122I; (6) S116V, W194Y; (7) K138V, W194Y; (8) I123P, F122I; (9) I123P, W194Y; (10) F122I, W194Y.
[0008] As a further optimization scheme of the present application, the single mutation site of the Sortase A transpeptidase mutant with higher thermostability than the 7+SrtA enzyme comprises at least one of K138I, K138V, S116V, I123P, F122I, D176P, Q178R, A135K, E85V, D82N, W194Y, K198L, D186N, S116A, E85Q, R151Y.
[0009] As a further optimization scheme of the present application, the single mutation site of the Sortase A transpeptidase mutant with higher activity than the 7+SrtA enzyme is I123P, and the double mutation site comprises at least one group of (1)-(9): (1) K138V, I123P; (2) K138V, S116V; (3) K138V, F122I; (4) S116V, I123P; (5) S116V, F122I; (6) S116V, W194Y; (7) K138V, W194Y; (8) I123P, F122I; (9) F122I, W194Y.
[0010] As a further optimization scheme of the present application, the double mutation site of the Sortase A transpeptidase mutant is K138V, I123P or K138V, S116V, and the protein sequence of the Sortase A transpeptidase mutant obtained by the above mutation is shown in SEQ ID NO. 3 or SEQ ID NO. 4.
[0011] As a second aspect of the present application, a polynucleotide encoding the Sortase A transpeptidase mutant according to any one of the above is also provided.
[0012] As a further optimization scheme of the present application, the polynucleotide sequence encoding the protein sequence of the Sortase A transpeptidase mutant shown in SEQ ID NO. 3-4 is shown in SEQ ID NO. 5-6, respectively.
[0013] As a third aspect of the present application, a recombinant plasmid, which is an expression vector containing the polynucleotide according to any one of the above and capable of translating and expressing the Sortase A transpeptidase mutant according to any one of the above, is also provided.
[0014] As a fourth aspect of the present application, the use of the 7+SrtA enzyme mutant according to any one of the above in the synthesis and modification of proteins is also provided.
[0015] As a fifth aspect of the present application, there is also provided an immobilized Sortase A transpeptidase mutant, which is obtained by fixing the Sortase A transpeptidase mutant according to any one of the above through thiol coupling to magnetic beads by using cysteine.
[0016] As a sixth aspect of the present application, there is also provided an application of the immobilized Sortase A transpeptidase mutant according to the above in protein labeling.
[0017] The present application has the following beneficial effects: The present application takes the 7+SrtA enzyme disclosed in the prior art as the research object, further designs and modifies the amino acid sequence thereof, provides a series of single-point mutations and double-point mutations for the 7+SrtA enzyme, and further screens two high-quality Sortase A transpeptidase mutants through heat stability and activity tests. The yield of the two high-quality Sortase A transpeptidase mutants is further improved compared with that of the 7+SrtA enzyme, the heat stability is improved by more than about 10℃, and the activity is improved by nearly 3 times. The two high-quality Sortase A transpeptidase mutants have higher yield, activity and heat stability than the 7+SrtA enzyme, have wider application conditions and stronger practical application value, and are more suitable for large-scale production and industrial use. BRIEF DESCRIPTION OF DRAWINGS
[0018] Figure 1 、 Figure 2 are the small-scale purification results of the 7+SrtA enzyme and the single-mutant protein thereof provided by the present application; Figure 3 are the small-scale purification results of the double-mutant protein of the 7+SrtA enzyme provided by the present application; Figure 4 are the activity detection results of the single-mutant protein of the 7+SrtA enzyme provided by the present application; Figure 5 are the activity detection results of the double-mutant protein of the 7+SrtA enzyme provided by the present application; Figure 6 are the affinity chromatography purification results of the 7+SrtA enzyme, the 7+SrtA (K138V, S116V) mutant protein and the 7+SrtA (K138V, I123P) mutant protein provided by the present application; Figure 7 are the LC-MS detection results of the protein modification function of the immobilized recombinant 7+SrtA enzyme provided by the present application; Figure 8 are the LC-MS detection results of the protein modification function of the immobilized recombinant 7+SrtA (K138V, S116V) mutant protein provided by the present application; Figure 9 LC-MS detection results of the immobilized recombinant 7+SrtA (K138V, I123P) mutant protein provided by the application for protein modification function. DETAILED DESCRIPTION
[0019] The following detailed description of the application is made with reference to the accompanying drawings, it is necessary to point out here that the following detailed description is only used to further illustrate the application, and cannot be understood as limiting the scope of protection of the application, and those skilled in the art can make some non-essential improvements and adjustments to the application according to the above application content.
[0020] 1. Materials and reagents The methods used in the application are conventional methods known to those skilled in the art, and if no specific conditions are specified, the conventional conditions or the conditions recommended by the manufacturer are used, and if no manufacturer of the reagents or instruments is specified, they are conventional products that can be obtained by market purchase.
[0021] 2. Methods 2.1. Construction of 7+SrtA enzyme and mutant protein plasmid The present application takes the 7+SrtA enzyme disclosed in the prior art as the research object, specifically, the construction of 7+SrtA enzyme is a truncation of 60-206 on the wild type Sortase A transpeptidase protein sequence, and further designs P94R, D160N, D165A, G167E, K190E, K196T, E105K, E108Q, D124G, Y187L, E189R mutation sites (the sequence number of the mutation site is numbered according to the position of the wild type Sortase A transpeptidase protein sequence), the protein sequence of 7+SrtA enzyme is SEQ ID NO. 2, and the gene sequence of 7+SrtA enzyme is obtained by gene synthesis method, the protein sequence of wild type Sortase A transpeptidase (1-206) (Uniprot: Q2FV99) is SEQ ID NO. 1.
[0022] All mutant proteins provided by the application are designed with corresponding mutant primers based on the protein sequence of 7+SrtA enzyme and constructed according to the method of molecular cloning, including K138I, K138V, S116V, I123P, F122I, D176P, Q178R, A135K, E85V, D82N, W194Y, K198L, D186N, S116A, E85Q, R151Y, A135N, R99K, V193K, T203K, N127D, T121R, D80A, D80S, T203E, a total of 25 single mutant proteins; 10 double mutant proteins of (K138V, I123P), (K138V, S116V), (K138V, F122I), (S116V, I123P), (S116V, F122I), (S116V, W194Y), (K138V, W194Y), (I123P, F122I), (I123P, W194Y), (F122I, W194Y).
[0023] It should be noted that the mutation sites of the single mutant protein and the double mutant protein are numbered according to the site position on the wild type Sortase A transpeptidase protein sequence.
[0024] 7. The SrtA enzyme and its mutant proteins are all constructed on the pET-29a vector (Kingsway), and the C-terminus of all 7+SrtA enzyme and its mutant proteins further carries a LE-6His tag sequence (where "LE" is a linker and 6His is a tag sequence composed of six histidines, used for affinity purification). The gene sequence of the constructed recombinant protein is verified correct by a sequencing company.
[0025] 2.2, Small-scale expression and purification of 7+SrtA enzyme and its mutant proteins Using conventional molecular biology methods, the above constructed 7+SrtA enzyme and 7+SrtA enzyme mutant protein plasmids are respectively transformed into BL21 (DE3) E. coli competent cells in a super-clean bench, and cultured at 37°C overnight. Single colonies of overnight culture are picked into 5 ml of LB liquid medium and cultured at 37°C. When the OD 600 of the bacterial solution is 0.6-0.8, a small amount of bacterial solution is fixed with loading buffer, and a small amount of bacterial solution is added to glycerol and frozen at -80°C. The remaining bacterial solution is added with 0.5 mM IPTG, and after 16 hours of induction at 15°C, the bacterial cells are collected and the induced bacterial solution is detected by SDS-PAGE.
[0026] The collected bacterial cells were dissolved in lysis buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5% glycerol), and then sonicated. After centrifugation at 12000 rpm for 10 minutes at 4 °C, the supernatant was collected. 50 μL of NiBestarose FF packing material pre-treated with lysis buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5% glycerol) was added to the supernatant, and the mixture was incubated at 4 °C for 30 minutes. The incubated sample was then centrifuged at 12000 rpm for 10 minutes at 4 °C. 1 mL of buffer was added, and the sample was washed three times. Then, 1 mL of washing buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5% glycerol, 20 mM imidazole) was added to wash away impurities. Finally, 100 μL of elution buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 5% glycerol, 20 mM imidazole) was added. Centrifuge at 12000 rpm and 4 ℃ for 5 minutes with glycerol (300 mM imidazole) and collect the eluted sample. For each step, a small amount of sample is retained and fixed with loading buffer and then analyzed by SDS-PAGE.
[0027] The experimental results are shown in Figure 1 and Figure 2 Of the 25 single-point mutations, all single mutant proteins were clearly expressed. Purification of all expressed samples revealed that all single mutant proteins could be eluted with high purity.
[0028] The small-scale test results for double mutant proteins were similar to those for single mutant proteins. All double mutant proteins could be eluted with high purity. The small-scale test results for double mutant proteins are shown below. Figure 3 .
[0029] 2.3 Detection of the thermal stability of mutant proteins The thermal stability test was performed using nano-differential scanning fluorescence (nanoDSF). The specific procedure is as follows: Take 20 μL of protein at a concentration of 0.5 mg / ml and add it to each well of a 384-well experimental plate. After shaking and centrifuging (to avoid sample inhomogeneity or air bubbles being introduced during sample aspiration), place the experimental plate on the sampling rack and use a Nano DSF capillary to aspirate the sample, ensuring the capillary is completely filled. Place the capillary in the nanoDSF instrument, set the initial temperature to 20 °C, and increase the temperature at a rate of 2.0 °C per minute until it reaches 90 °C. The instrument will perform the temperature increase according to the set parameters and monitor in real time.
[0030] The specific numerical results of the Tm values of all single mutant proteins are shown in Table 1.
[0031] Table 1, Tm values of single mutant proteins ; As shown in Table 1, 16 single mutant proteins that can improve the thermal stability of 7+SrtA enzyme were screened from 25 single mutant proteins, including K138I, K138V, S116V, I123P, F122I, D176P, Q178R, A135K, E85V, D82N, W194Y, K198L, D186N, S116A, E85Q, and R151Y, with Tm values ranging from 44.26-50.77℃. Among them, the Tm values of K138I, K138V, S116V, I123P, and F122I increased by 2.57-6.66℃ compared with 7+SrtA enzyme (44.11℃).
[0032] After combining the single mutant sites with significantly improved Tm values, 10 double mutant proteins were obtained according to the small-scale expression scheme of single mutant proteins. The thermal stability of the double mutant proteins was detected according to the detection steps described above, and the Tm values of the double mutant proteins detected are shown in Table 2.
[0033] Table 2, Tm values of double mutant proteins ; As can be seen from Table 2, the Tm values of all double mutants are 2.85-11.74℃ higher than that of 7+SrtA enzyme, among which the effects of (K138V, I123P) and (K138V, S116V) are the best, and the Tm values of the corresponding double mutant proteins are increased by more than 10℃.
[0034] 2.4, Activity detection of 7+SrtA enzyme and its mutant proteins Sortase A as a transpeptidase, can specifically recognize Leu-Pro-X-Thr-Gly (LPXTG) polypeptide sequence, and cut between the specific site threonine Thr and glycine Gly residues, the resulting threonine carboxyl terminal can be covalently linked to the target. The present application takes Abz-LPETGK (Dnp)-NH2 as the substrate (purchased from Shanghai Jetnai Biological Technology Co., Ltd.), wherein Abz (Ortho-aminobenzoic acid) is used as a fluorescent group, and Dnp (2,4-Dinitrophenyl) is used as a quenching group. When the polypeptide substrate sequence is complete, it shows internal fluorescence quenching. However, when Sortase A enzyme cuts between the Thr and Gly residues of the substrate, the fluorescent group Abz and the quenching group Dnp are separated, releasing the Abz fluorescence signal and can be continuously detected. Therefore, the principle can be used for quantitative analysis of the enzyme activity of Sortase A, and the specific steps are as follows: Prepare buffer solution: 50 mM HEPES pH 7.5, 150 mM NaCl, substrate is 50 μM Abz / Dnp, reaction temperature is room temperature, and the 7+SrtA protein obtained in step 2.2 is diluted to 100 μM with the buffer solution. The prepared 7+SrtA sample and substrate are added to a 384-well plate and set up a duplicate well, and then centrifuged and shaken to mix immediately. The fluorescence signal value generated after the substrate is cut is collected using a TECAN Spark enzyme marker. The data is analyzed using GraphPad Prism9 software, and finally the enzyme activity parameters of the proteinase to be tested are obtained.
[0035] Among them, the activity results of the single mutant protein are as shown in Figure 4 Among them, the activity results of the single mutant protein are as shown in
[0036] The activity data of the double mutant protein are shown in Figure 5 , except that the activity of the (I123P, W194Y) double mutant protein is 31 % lower than that of 7+SrtA, the activities of the other nine double mutant proteins are increased by 121 %-349 %, which have high activity. Among them, the (K138V, I123P) double mutant protein and the (K138V, S116V) double mutant protein have a larger activity increase of 284 % and 349 % respectively, indicating that these mutant proteins with improved thermal stability still maintain good activity.
[0037] 2.5, Expression and purification of high-quality mutant protein of 7+SrtA enzyme To further study the function of 7+SrtA enzyme high-quality mutant protein, two (K138V, I123P) and (K138V, S116V) double mutant proteins with significantly improved Tm value and activity were selected, i.e. 7+SrtA (K138V, I123P) mutant protein and 7+SrtA (K138V, S116V) mutant protein, the amino acid sequences of which are shown in SEQ ID NO. 3 and SEQ ID NO. 4, and the nucleotide sequences of which are shown in SEQ ID NO. 5 and SEQ ID NO. 6.
[0038] To improve the yield of mutant protein, a 6His-SUMO tag sequence was further added to the N terminus of the sequence shown in SEQ ID NO. 3 and SEQ ID NO. 4, and a cysteine C was added to the C terminus of the sequence as a linker with “LE”, to construct 6His-SUMO-7+SrtA (K138V, I123P)-LEC and 6His-SUMO-7+SrtA (K138V, S116V)-LEC plasmids. The amino acid sequences of the two 7+SrtA mutant proteins after the above sequence design are shown in SEQ ID NO. 3 and SEQ ID NO. 4, and the 6His-SUMO tag sequence is shown in SEQ ID NO. 7.
[0039] To compare the application effect of 7+SrtA enzyme in the subsequent comparison, 6His-SUMO-7+SrtA-LEC protein of the same form was also constructed. The amino acid sequence of 6His-SUMO-7+SrtA-LEC protein is shown in SEQ ID NO. 8. The 7+SrtA enzyme and its mutant proteins were constructed in pET-29a vector, and the gene sequences of the constructed recombinant proteins were verified correct by a sequencing company.
[0040] 2.5.1, Purification of 7+SrtA enzyme high-quality mutant protein Using conventional molecular biology methods, the 7+SrtA enzyme and its mutant plasmids constructed above were transformed into BL21 (DE3) E. coli competent cells in a super-clean bench, the strains were inoculated into 50 ml of LB liquid medium and cultured at 37 °C overnight, the bacteria cultured overnight were inoculated into 1 L of LB liquid medium at a ratio of 1:100, and the bacteria were cultured at 37 °C until the OD 600 was 0.6-0.8, 0.5 mM IPTG was added, and the bacteria were cultured at 15 °C overnight. The bacteria were collected by centrifugation at 5000 rpm for purification.
[0041] The specific purification steps are as follows: the collected bacteria were weighed, and the corresponding volume of buffer A (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol) was added according to the ratio of 1:10, the bacteria were broken by using high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation at 16000 rpm. 5 mL of Ni Bestarose FF pre-packed affinity column was first washed with buffer for 10 column volumes, then the lysate supernatant was loaded onto the column, and the column was washed with buffer for 5 column volumes to remove the proteins not bound to the column. Different gradient imidazole solutions were used to elute the proteins. Since the 7+SrtA enzyme and its mutants have SUMO enzyme cleavage sites, the target protein and SUMO protease were mixed and incubated overnight for enzyme cleavage. Then the mixture was loaded onto the Ni Bestarose FF affinity column, and different gradient imidazole solutions were used to elute the proteins on the column. The 7+SrtA enzyme with the removed tag cannot be bound to the column because it does not have a His tag, so it will penetrate out. The penetrated protein was collected, subjected to SDS-PAGE detection, and the protein concentration was determined by Nanodrop. The protein yield was calculated.
[0042] According to the SDS-PAGE results (see Figure 6 ), after purification, the yield of 7+SrtA (K138V, S116V) mutant protein was 25.50 mg / L, the yield of 7+SrtA (K138V, I123P) mutant protein was 12.24 mg / L, and the yield of 7+SrtA enzyme was 10.04 mg / L. After affinity purification, the yields of 7+SrtA (K138V, S116V) mutant protein and 7+SrtA (K138V, I123P) mutant protein were 2.5 times and 1.2 times that of 7+SrtA enzyme, respectively. In addition, in terms of purity, the purities of the two 7+SrtA enzyme high-quality mutant proteins were also significantly improved compared with that of 7+SrtA enzyme.
[0043] 2.6, Immobilization of 7+SrtA enzyme and its mutant proteins Take 100 μl of thiol-coupled magnetic beads and separate the magnetic beads and buffer on a magnetic adsorption rack, wash 3 times with 2 volumes of buffer (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 5% glycerol), then add 3 mg of 7+SrtA enzyme and its mutant protein mixture, incubate at 4°C overnight, then wash with 3 volumes of buffer, collect the supernatant, measure the A280 value, add 50 mM cysteine blocking buffer and incubate at 4°C for 2 hours, and finally wash with 3 volumes of buffer. Calculate the amount of enzyme remaining after coupling. The results show that the remaining amounts of 7+SrtA enzyme, 7+SrtA (K138V, S116V) mutant protein and 7+SrtA (K138V, I123P) mutant protein after incubation are 1.37 mg, 0.6 mg and 2.2 mg, respectively. Therefore, the immobilization amounts of the three enzymes on the magnetic beads are calculated to be 16.30 mg / ml, 24.00 mg / ml and 8.00 mg / ml, respectively. It can be seen that the immobilization amount of 7+SrtA (K138V, S116V) mutant protein is higher than that of 7+SrtA enzyme, while the immobilization amount of 7+SrtA (K138V, I123P) mutant protein is lower than that of 7+SrtA enzyme.
[0044] 2.7, verification of immobilized 7+SrtA enzyme function Prepare immobilized 7+SrtA enzyme and its mutant protein, as well as target protein 1 and modifier that need to be modified. The molecular weight of target protein 1 is 97398.5 Da, and the sequence of target protein 1 is shown in SEQ ID NO. 9. The modifier is Biotin-LPET-G (2-hydroxyacetic acid)-GH (Biotin) (Beijing Zhongke Yaguang Biotechnology Co., Ltd.), and its molecular weight is 666 Da. Mix the immobilized 7+SrtA enzyme or 7+SrtA enzyme mutant protein, target protein and modification protein according to the mass ratio of 1:300:10, and react at 4°C for 2 hours, 4 hours and 12 hours, respectively. Use LC-MS to detect the molecular weight of the sample in the reaction system before and after reaction to determine whether the 7+SrtA enzyme and its mutant protein have labeled the modifier on the target protein 1. The results are shown in Figure 7 、 Figure 8 、 Figure 9
[0045] Result analysis: According to the range of LC-MS detection, it can be seen that after 2 h of reaction, 7+SrtA enzyme, 7+SrtA (K138V, I123P) mutant protein and 7+SrtA (K138V, S116V) mutant protein respectively labeled 30%, 80% and 100%, it can be seen that 7+SrtA (K138V, S116V) mutant protein completely labeled the modifier on the target protein 1 after 2 h of reaction, indicating that the activity of 7+SrtA (K138V, S116V) mutant protein is higher; 7+SrtA enzyme, 7+SrtA (K138V, I123P) mutant protein and 7+SrtA (K138V, S116V) mutant protein, all show complete labeling after 4 h of reaction, and after 12 h of reaction, 7+SrtA enzyme shows 20% reverse reaction, 7+SrtA (K138V, S116V) mutant protein has no reverse reaction, and 7+SrtA (K138V, I123P) mutant protein shows 1% reverse reaction, indicating that 7+SrtA (K138V, S116V) mutant protein has high activity and is not prone to hydrolysis reaction, and is more suitable for protein modification.
[0046] 3、Conclusion The above shows that the Sortase A transpeptidase mutant protein provided by the application has higher protein yield, higher enzyme activity and better thermal stability, has wider application conditions and stronger practical application value, and is more suitable for large-scale production and industrial use.
[0047] The above-described embodiments only express several embodiments of the application, which are described in more detail and in detail, but should not be understood as limiting the scope of the patent of the application. It should be noted that for ordinary skilled in the art, without departing from the concept of the application, a number of modifications and improvements can be made, which are within the scope of protection of the application.
Claims
1. A Sortase A transpeptidase mutant, characterized in that, The Sortase A transpeptidase mutant is obtained by single or double mutation based on the protein sequence shown in SEQ ID NO.
2. The protein sequence shown in SEQ ID NO.2 is a truncated mutant of positions 60-206 on the wild-type Sortase A transpeptidase shown in SEQ ID NO.
1. Based on the sequence of the wild-type Sortase A transpeptidase as shown in SEQ ID NO.1, the single mutation site of the Sortase A transpeptidase mutant includes at least one of K138I, K138V, S116V, I123P, F122I, D176P, Q178R, A135K, E85V, D82N, W194Y, K198L, D186N, S116A, E85Q, R151Y, A135N, R99K, V193K, T203K, N127D, T121R, D80A, D80S or T203E; The double mutation sites of the Sortase A transpeptidase mutant include at least one set of (1)-(10): (1) K138V, I123P; (2) K138V, S116V; (3) K138V, F122I; (4) S116V, I123P; (5) S116V, F122I; (6) S116V, W194Y; (7) K138V, W194Y; (8) I123P, F122I; (9) I123P, W194Y; (10) F122I, W194Y.
2. The Sortase A transpeptidase mutant according to claim 1, characterized in that, The single mutation site includes at least one of K138I, K138V, S116V, I123P, F122I, D176P, Q178R, A135K, E85V, D82N, W194Y, K198L, D186N, S116A, E85Q, and R151Y.
3. A Sortase A transpeptidase mutant according to claim 1, characterized in that, The double mutation sites are at least one of (1)-(9): (1) K138V, I123P; (2) K138V, S116V; (3) K138V, F122I; (4) S116V, I123P; (5) S116V, F122I; (6) S116V, W194Y; (7) K138V, W194Y; (8) I123P, F122I; (9) F122I, W194Y.
4. A Sortase A transpeptidase mutant according to claim 1, characterized in that, The double mutation sites of the Sortase A transpeptidase mutant are K138V, I123P or K138V, S116V, and the protein sequence of the Sortase A transpeptidase mutant obtained by the above mutation is shown in SEQ ID NO.3 or SEQ ID NO.
4.
5. A polynucleotide, characterized in that, The polynucleotide encodes the Sortase A transpeptidase mutant as described in any one of claims 1-3.
6. A polynucleotide according to claim 5, characterized in that, The polynucleotide sequences encoding the protein sequences of the Sortase A transpeptidase mutants shown in SEQ ID NO.3-4 are shown in SEQ ID NO.5-6, respectively.
7. A recombinant plasmid, characterized in that, The recombinant plasmid is an expression vector containing the polynucleotides as described in any one of claims 5-6 and capable of translating and expressing the Sortase A transpeptidase mutant as described in any one of claims 1-4.
8. The application of the Sortase A transpeptidase mutant as described in any one of claims 1-4 in protein synthesis and modification.
9. An immobilized Sortase A transpeptidase mutant, characterized in that, The immobilized Sortase A transpeptidase mutant is obtained by immobilizing the Sortase A transpeptidase mutant as described in any one of claims 1-4 onto magnetic beads using cysteine via thiol coupling.
10. The application of the immobilized Sortase A transpeptidase mutant as described in claim 9 in protein labeling.