CN116144687B - Acid protease AGP and its preparation method and application - Google Patents

Abstract

The application provides acid protease AGP and its preparation method and application. The method for preparing acid protease AGP comprises: inserting the target gene into an expression vector, constructing a recombinant plasmid; after the recombinant plasmid is transformed into a competent cell, adding isopropylthiogalactopyranoside (IPTG) to induce expression; The acid protease AGP was purified from the induced protein. The acidic proteolytic enzyme AGP provided by this application has the following characteristics: it still maintains relatively strong activity under acidic conditions (pH 2.5) and low temperature, and a small amount of denaturant and reducing agent; The sites are aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), histidine (His), lysine (Lys), etc., which are highly complementary to the Pepsin restriction site The peptide length produced by AGP digestion is shorter than that produced by Pepsin.

Description

Acid protease AGP and its preparation method and application

technical field

The invention relates to the technical field of biological mass spectrometry, more specifically, to acid protease AGP and its preparation method and application.

Background technique

Protein is the foundation of life, and all manifestations of life are essentially the embodiment of protein functions. Understanding the structure and dynamic changes of proteins in cells, tissues, and even the entire living body will give you a comprehensive understanding of the occurrence and development of diseases, grasp the key to disease diagnosis and treatment, and improve the efficiency of drug development.

Hydrogen Deuterium Exchange Mass spectrometry (HDX-MS) is a common method for studying protein structure. This technology uses mass spectrometry to monitor the exchange characteristics of amide hydrogen and deuterium water in the protein backbone, reflecting the conformational characteristics and characteristics of proteins in solution. Dynamic changes. HDX-MS has been widely used in membrane protein interaction, antibody, and even protein assembly and virus research due to its advantages of rapid analysis, sensitivity, low sample consumption, and suitability for analysis of complex protein systems. Since HDX-MS is mainly based on the bottom-up (Bottom up) proteomics method, it is necessary to use protease digestion to generate peptides before MS analysis. In order to minimize the backcrossing of deuterium and hydrogen on the surface of the deuterium-labeled protein, the protein samples were incubated in the deuterated solution for a certain period of time, and then the reaction was terminated at pH 2.5 and 0 °C. In general, quenching solution was added to the experiment and adjusted to pH 2.5, while the temperature was controlled at 0°C to minimize the HDX backcrossing rate. However, according to the properties of different protein samples, different concentrations of denaturant (urea or guanidine hydrochloride) and reducing agent (TCEP) are added to the quenching solution to help protein denaturation and reduction, thereby improving the effect of enzyme digestion and sequence coverage. To prevent backcrossing in subsequent analyses, all steps after quenching should be done as soon as possible at pH 2.5 and at low temperature, and the protein should be cleaved into peptides using an acidic protease such as pepsin. In general, proteases used in HDX-MS must meet the following key criteria, first, maintain activity at pH 2.5 and low temperature; second, have a wide range of enzyme cleavage sites to maximize protein sequence coverage and sequence coverage redundancy. In addition, a certain degree of compatibility with denaturants and reducing agents can effectively expand the use scenarios of proteases. However, the types of proteases that can function under such harsh conditions are very limited.

Pepsin is the most commonly used acid proteolytic enzyme for HDX-MS. Under typical HDX-MS experimental conditions, the main restriction sites (P1 position) of pepsin are Met, Leu and Phe; and when His, Lys, Arg or Pro occupy the P1 position, pepsin enzymatic activity is inhibited. Therefore, pepsin cannot fully digest disordered proteins or disordered regions of protein structures (usually containing Pro and charged amino acids), thus affecting the acquisition of disordered protein or region structure information that is important for regulating protein biological functions. In addition, the average length of the peptides produced by Pepsin digestion was at the level of ~14 amino acids, and the reduction of the average length of the peptides was conducive to improving the structural resolution of the HDX-MS method. Based on this, the development of acidic proteolytic enzymes suitable for HDX-MS experiments is a very important topic.

Contents of the invention

The application provides an acidic proteolytic enzyme that can be used for HDX-MS analysis. The acidic protease is Aspergilloglutamic peptidase (AGP) from Aspergillus niger var.macrosporus, which is insensitive to aspartic acid type acidic endopeptidase.

The application provides a method for preparing acid protease AGP, comprising: inserting the target gene SEQ.ID.NO.1 into an expression vector to construct a recombinant plasmid; after transforming the recombinant plasmid into competent cells, adding isopropyl Induced expression with thiogalactoside (IPTG); acid protease AGP was purified from the induced expressed protein.

In some embodiments, the conditions for inducing expression are: when the recombinant expression host bacteria are shaken and cultured at 37°C until the OD600 is 0.6, isopropylthiogalactoside with a final concentration of 1 mM is added, and shaken at 37°C for 4 hours .

In some embodiments, the purification conditions to obtain the acid protease AGP are as follows: elution with imidazole content of 0mM, 10mM, and 30mM for 4 times respectively, after washing away impurities, and elution with an elution buffer with an imidazole content of 300mM target protein.

The present application also provides the acid protease AGP obtained by the above method.

The application also provides the application of acid proteolytic enzyme AGP in HDX-MS and proteomics.

The acidic proteolytic enzyme AGP of the present application has the following characteristics: it still maintains relatively strong activity under acidic conditions (pH 2.5) and low temperature, and a small amount of denaturant and reducing agent; The points are aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), histidine (His), lysine (Lys), etc., which are highly complementary to the Pepsin restriction site ; The length of the peptide produced by AGP digestion is shorter than that of Pepsin.

Description of drawings

Figure 1 shows the SDS-PAGE and Western Blot diagrams of the expressed and purified protease AGP.

Figure 2 shows the activity of protease AGP to digest standard peptides under different conditions of pH, temperature, denaturant and reducing agent.

Figure 3 shows the overlap of digested peptides from proteases AGP and Pepsin when digesting a standard protein mixture.

Figure 4 shows the boxplots of lengths of digested peptides from proteases AGP and Pepsin when the standard protein mixture is digested.

Figure 5 shows the mass distribution and boxplot of digested peptides from proteases AGP and Pepsin when the standard protein mixture is digested.

Fig. 6 shows a histogram comparing the ratios of enzyme cleavage sites of protease AGP and Pepsin.

Detailed ways

The following examples can enable those skilled in the art to understand the present invention more fully, but do not limit the present invention in any way.

The present application provides an acidic proteolytic enzyme AGP, which is derived from Aspergillus nigervar. macrosporus. The beneficial effect is that the enzyme has a clear preference for enzymatic cleavage of charged amino acid sites, is highly complementary to the pepsin cleavage site, and the average length of the generated peptides is shorter than that of pepsin, and the enzyme can be used for HDX-MS experiments are used for protein structure analysis as well as proteomics analysis.

The acid proteolytic enzyme AGP provided by the invention comes from recombinant expression.

1. The expression steps of AGP

The gene sequence of the target gene SEQ.ID.NO.1 (SEQ.ID.NO.1 is: Atgggtcaccatcaccatcaccatgtcggactcagaagtcaatcaagaagctaagccagaggtcaagccagaagtcaagcctgagactcacatcaatttaaaggtgtccgatggatcttcagagatcttcttcaagatcaaaaaagaccactcctttaagaagg ctgatggaagcgttcgctaaaagacagggtaaggaaatggactccttaagattcttgtacgacggtattagaattcaagctgatcagacccctgaagatttggacatggaggataacgatattattgaggctcacagagaacagattggtggccaaGGATCCGCGCCGCTGACCGAAAAACGCCGCGCGCGCAAAGAAGCGCGCGGCGGGCAAACG CCATAGCAACCCGCCGTATATTCCGGGCAGCGATAAAGAAATTCTGAAACTGAACGGCACCAGCAACGAAGATTATAGCAGCAACTGGGCGGGCGCGGTGCTGATTGGCGATGGCTATACCAAAGTGACCGGCGAATTTACCGTGCCGAGCGTGAGCGCGGCAGCAGCAGCAGCAGCGGCTATGGCGGCGGCTATGGCTATTATAAAACAAAC GCCAGAGCGAAGAATATTGCGCGAGCGCGTGGGTGGGCATTGATGGCGATACCTGCGAAACCGCGATTCTGCAGACCGGCGTGGATTTTTGCTATGAAGATGGCCAGACCAGCTATGATGCGTGGTATGAATGGTATCCGGATTATGCGTATGATTTTAACGATATTACCATTAGCGAAGGCGATACCATTAAAGTGACCGTGGAAGCGACCAGCAAAAGCAGC GGCAGCGCGACCGTGGAAAACCTGACCACCGGCCAGAGCGTGACCCATACCTTTAGCGGCAACGTGGAAGGCGATCTGTGCGAAACCAACGCGGAATGGATTGTGGAAGATTTTGAAAGCGGCGATAGCCTGGTGGCGTTTGCGGATTTTGGCAGCGTGACCTTTACCAACGCGGAAGCGACCAGCGATGGCAGCACCGTGGGCCCGAGCGATGCGA CCGTGATGGATATTGAACAGGATGGCACCGTGCTGACCGAAACCAGCGTGAGCGGCGATAGCGTGACCGTGACCTATGTGTAACTCGAG) was inserted into the PATX-SUMO expression vector (cloning strategy: BamHI/XhoI) to construct a recombinant plasmid. After using this plasmid to transform BL21 competent cells, add isopropylthiogalactopyranoside (IPTG) to induce expression. After a series of conditional explorations, the expression conditions were finally determined: the recombinant expression host bacteria were shaken and cultured at 37°C to an OD600 of about When it was 0.6, IPTG with a final concentration of 1 mM was added, and cultured with shaking at 37°C for 4h. Nickel magnetic beads were used to extract and purify the protease AGP, and the purification conditions were determined to be eluted four times with imidazole content of 0mM, 10mM, and 30mM respectively. The harvested proteins were identified by SDS-PAGE and Western Blot.

(1) SDS-PAGE and Western Blot identification method

Take a protease sample, add 5×SDS-PAGE loading buffer at a volume ratio of 1:4, mix well, and heat at 95°C for 5 minutes to denature the protein. Prepare SDS-PAGE gel for loading. Before the sample enters the gel, the voltage is controlled at 100-200V. When the bromophenol blue indicator in the sample reaches the separation gel, the voltage is raised to 200V, and the voltage is kept stable during the electrophoresis process. After electrophoresis, the gel was taken out, and Coomassie brilliant blue staining solution was added and shaken slowly for 1 h. Pour off the dye solution, wash with distilled water, add an appropriate amount of decolorization solution, and shake quickly to elute until the bands are clear. Then carry out Western Blot identification. The protein was transferred to polyvinylidene fluoride (PVDF) membrane by electrophoresis. Electroporation on ice, 230mA constant current electroporation for 80min, voltage kept above 90V. After electrotransfer, soak the PVDF transfer membrane with 5% milk at room temperature, and shake slowly for 1 hour to seal the pores. Then wash the membrane with 1 × TBST solution for 4 times, 5 min each time. Place the membrane in His-tag antibody diluent and incubate overnight at 4°C with rotation. After the incubation, the PVDF membrane was taken out, and the membrane was quickly shaken and washed 4 times with 1×TBST solution, 5 min each time. Place the membrane in the diluent of the mouse secondary antibody, incubate with rotation at room temperature for 1 h, then wash the membrane with 1× TBST solution for 4 times, each time for 5 min, and develop.

(2) Results

The results of Coomassie brilliant blue staining are shown in A in Figure 1, and the results of Western Blot are shown in B in Figure 1. The band of protease AGP can be observed at a molecular weight of 50 kDa.

2. Investigation of AGP digestion conditions

(1) enzyme digestion method

AGP and the standard peptide Substance P (RPKPQQFFGLM) were incubated at a ratio of 1:10 (W/W) at pH 1, pH 2.5, pH 5, and pH 7 at 37°C for 15 minutes. The reaction products were determined by LC-MS method.

(2) Results

The results are shown in a in Figure 2. Under acidic conditions of pH 1-2.5, the detected substances are mainly the hydrolyzed fragment RPKPQQFF of SubstanceP, indicating that AGP is active under acidic conditions; and when pH ≥ 4, The detected substances are mainly Substance P itself, and the activity of AGP drops significantly at this time. The same trend was also observed at 0 °C except at room temperature. In order to activate AGP activity and obtain better digestion efficiency at low temperature, AGP was equilibrated at room temperature for 10 min before adding the substrate. Compared with 0°C, the activity increased by about 46% at pH=2.5. The quenching step in HDX-MS experiments usually involves the use of denaturants and reducing agents. Therefore, based on the enzyme activity test, the HDX MS experiment conditions, namely 0 °C and acidic pH (2.5) conditions, were further investigated. AGP activity levels at increasing concentrations of urea or denaturing agents (TCEP, trichloroethyl phosphate). As shown in b in Figure 2, when the concentration of urea (Urea) increased to 1.5M, AGP still retained about 50% of its activity. As shown in c in Fig. 2, when the concentration of TCEP was increased to 200 mM, the AGP activity remained about 40%.

3. Comparison of proteolytic enzymes AGP and Pepsin digestion

(1) enzyme digestion method

Take 5ug of the standard protein mixture (Table 1), add urea and 10mM dithiothreitol (DTT) at a final concentration of 8M, and incubate at 37°C for 2h to denature the protein and open the disulfide bond. After the reaction was completed, indole-3-acetic acid (IAA) with a final concentration of 20 mM was added to the reaction system, and the disulfide bond was blocked by reacting for 40 min at room temperature and protected from light. After completion of the reaction, add 7 times the volume of buffer to dilute the urea concentration to 1M. The buffer used by proteolytic enzyme AGP is 0.2M glycine-hydrochloric acid buffer (pH 2.2), and the buffer used by pepsin is 10mM HCl (pH 2.0). Adjust the pH of the protein solution to 2-2.5, add AGP or pepsin according to the ratio of enzyme to protein of 1:20 (w/w), react at 37°C for 6 hours, and heat to terminate the reaction after the reaction is completed. The samples were analyzed by LC-MS after desalting, and each sample was injected three times. Table 1 shows standard protein mix information.

Table 1

(2) Results

The results are shown in Figure 3. In the three repeated injections, AGP digestion produced a total of 1264 peptides with 3 repeats, and pepsin produced 1306 peptides with 3 repeats. Only 149 peptides were found by comparison. The segments coincide, indicating that the AGP restriction site is highly complementary to pepsin. In addition, the peptides produced by AGP digestion are more distributed within m/z 2000, and the median length of the average peptide is 12 (m/z 1383.20); while the peptides produced by pepsin digestion are more distributed in m/z z1000-3000, the median length of the average peptide segment is 14 (m/z 1603.82) (Figure 4-Figure 5). Therefore, AGP can produce shorter peptides than pepsin, which can achieve higher structural resolution for HDX-MS-based protein structure analysis.

As shown in Figure 6, AGP has a wide range of enzyme cutting sites, and preferentially cuts the charged amino acid types on the P1 site, including His (11.74%), Lys (9.41%), Glu (14.14%), Asp ( 11.66%) and Phe (10.05%); while pepsin preferentially cuts Leu (25.66%), Phe (12.83%) and Glu (12.99%), and pepsin hardly cuts at His site (0.23%). In addition, the restriction ratios of AGP at Asn (6.51%), Gln (5.31%), and Arg (4.10%) sites were significantly higher than those of pepsin (corresponding to 1.32%, 1.63%, 2.02, and 0.70%). It can be seen that the cleavage sites of AGP and Pepsin are complementary, and can be used to obtain structural information of disordered proteins or disordered regions of proteins.

Therefore, the proteolytic enzyme AGP of the present application still maintains high activity under the conditions of pH 2.5, low temperature, and the presence of an appropriate amount of denaturant and reducing agent, and has a wide range of enzyme cleavage sites, the main enzyme cleavage sites are His and Lys , Glu, etc., are highly complementary to the pepsin cleavage site, and the resulting peptides are shorter than pepsin cleavage peptides, which can be used for protease cleavage and routine proteomics under HDX-MS experimental conditions enzyme cleavage reaction.

Those skilled in the art should understand that the above embodiments are only exemplary embodiments, and various changes, substitutions and changes can be made without departing from the spirit and scope of the present invention.

Claims (3)

1. an application of acid protease AGP in hydrogen-deuterium exchange mass spectrometry (HDX-MS), is characterized in that, the preparation method of acid protease AGP comprises: Insert the target gene SEQ.ID.NO.1 into the expression vector to construct a recombinant plasmid; After the recombinant plasmid is transformed into competent cells, adding isopropylthiogalactopyranoside (IPTG) to induce expression; Acid protease AGP was purified from the induced protein. 2. application according to claim 1, is characterized in that, the condition of described induced expression is: When the recombinant expression host bacteria were cultured with shaking at 37°C until the OD600 was 0.6, isopropylthiogalactopyranoside at a final concentration of 1 mM was added and cultured with shaking at 37°C for 4 hours. 3. application according to claim 1, is characterized in that, the purifying condition that purifying obtains acid protease AGP is: successively use the elution buffer solution of 0mM, 10mM, 30mM with imidazole content to wash out 4 times respectively, wash away miscellaneous protein Finally, the target protein was eluted with an elution buffer with an imidazole content of 300 mM.