Prokaryotic expression-based quantitative analysis method for protein binding metal ions

Abstract

The invention discloses a protein binding metal ion quantitative analysis method based on prokaryotic expression, relates to a protein and metal ion binding analysis method, and aims to solve the problems that the current protein binding metal ion analysis cost is high, the process is tedious, and metal ions cannot be quantitatively analyzed by most analysis methods. The method comprises the steps of target protein prokaryotic expression, purification and metal ion binding quantitative analysis. The method utilizes a label purification system, indirectly quantifies protein binding metal ions by detecting the content of metal ions in supernatant, and has the advantages of simplicity and convenience in operation, low cost, high sensitivity, strong universality and the like. The method is suitable for quantitative analysis of various metal ions and protein types, and provides an effective tool for protein function research and application. The method is applied to the field of biology.

Description

Technical Field

[0001] This invention relates to a quantitative analysis method for protein-bound metal ions based on prokaryotic expression. Background Technology

[0002] The interaction between metal ions and proteins plays a crucial role in many biological processes, such as the catalytic activity of metalloenzymes, the role of metal ions in cell signal transduction, and the influence of metal ions on protein folding and stability. Therefore, studying and quantitatively analyzing the binding of proteins to metal ions not only helps to understand the molecular mechanisms of protein function but also has important implications for disease diagnosis, drug development, and environmental monitoring.

[0003] Currently, common analytical methods for protein-metal ion binding include spectrometry, mass spectrometry, fluorescence spectrometry, and chromatography. Each method has its own advantages and disadvantages, and its applicable scope varies. Spectrometry is widely used to determine the concentration of metal ions and analyze the binding of metal ions to proteins. This method is simple to operate, but its sensitivity and selectivity are poor for low concentrations of metal ions and complex sample systems, and it is easily affected by interference. Mass spectrometry can provide detailed information about proteins and their metal ion binding sites. It has high sensitivity and high resolution, but it is complex to operate, expensive, and usually requires high-precision instruments. Fluorescent probe methods can specifically detect the binding of proteins to metal ions. For example, the emission intensity of certain fluorescent molecules changes when they bind to metal ions, so the metal-binding characteristics of proteins can be analyzed by measuring the intensity of the fluorescence signal. Fluorescence methods have high sensitivity and good spatial resolution, but require the selection of appropriate fluorescent probes. Chromatographic methods, such as high-performance liquid chromatography (HPLC), can separate protein-metal ion complexes and quantitatively analyze their binding by detecting peak area and retention time. Chromatography has high precision, but requires a long analysis time.

[0004] While existing analytical methods have met the needs of protein-metal ion binding analysis to some extent, they still have limitations in terms of cost and operational procedures. Therefore, developing a novel, more efficient, and accurate in vitro quantitative analytical method for protein-metal ion binding is of particular importance. Summary of the Invention

[0005] The purpose of this invention is to solve the problems of high cost, cumbersome process and inability of most analytical methods to quantitatively analyze metal ions in protein-bound metal ion analysis, and to provide a quantitative analysis method for protein-bound metal ions based on prokaryotic expression.

[0006] The present invention provides a quantitative analysis method for protein-bound metal ions based on prokaryotic expression:

[0007] I. Prokaryotic expression of the target protein: The ORF sequence of the target protein is inserted into a prokaryotic expression vector to construct a recombinant plasmid, which is then transformed into competent cells to obtain a prokaryotic expression strain, which is then induced to express the protein.

[0008] II. Purification of the target protein: Collect the bacterial cells obtained in step one, break them up, and then purify the fusion protein to obtain the purified fusion protein;

[0009] III. Quantitative analysis of protein-bound metal ions: The purified fusion protein was incubated with metal ions, purified again, and the supernatant was collected. The final content of metal ions was detected to indirectly quantify protein-bound metal ions.

[0010] The beneficial effects of this invention are:

[0011] This invention provides a method for quantitative analysis of protein-bound metal ions based on prokaryotic expression, which can be used for in vitro analysis of the content of metal ions bound to target proteins. This invention utilizes the specific binding of the target protein to metal ions, and through prokaryotic expression and a tag protein affinity purification system, detects the metal ion content in the supernatant of the system, thereby indirectly quantifying the metal ions bound to the protein. This method is simple to operate, has low requirements on protein sample concentration and metal ion sample type, and only requires an ELISA reader to complete the quantitative analysis of metal ions. Traditional methods are often limited to interactions between specific types of metal ions or proteins, while the analytical method of this invention is applicable to various metal ions and different types of protein systems, especially suitable for studying the interaction between metal ions and proteins in complex biological systems. Therefore, it has broad application prospects in multiple fields such as biomedicine, environmental protection, public health, and food safety.

[0012] This invention uses the calcium ion binding of *Populus simonii* PsnPLC as an example. A GST-PLC prokaryotic expression vector was constructed. After IPTG induction, bacterial cells were lysed, centrifuged, and the supernatant was collected and incubated with GST-tagged protein purification resin to purify the target protein. Subsequently, the GST-PsnPLC fusion protein was eluted with reduced glutathione. The fusion protein was then incubated with calcium ions again with GST-tagged protein purification resin, and the supernatant was collected. The calcium content in the supernatant was determined using a calcium ion detection kit, thereby determining the calcium ion binding content of GST-PsnPLC. Furthermore, the fusion protein lacking the EF chiral domain and C2 domain still possesses the ability to bind calcium ions. Attached Figure Description

[0013] Figure 1 A schematic diagram showing the complete PsnPLC sequence, the missing EF hand-shaped structural domain, and the missing C2 structural domain;

[0014] Figure 2The image shows the binding effect of PsnPLC with calcium ions analyzed by SDS-PAGE.

[0015] Figure 3 Image showing the binding effect of PsnPLC with calcium ions after SDS-PAGE analysis of the deletion of the EF chiral domain.

[0016] Figure 4 Image showing the binding effect of PsnPLC with calcium ions after the deletion of the C2 domain, as analyzed by SDS-PAGE.

[0017] Figure 5 The graph shows the effect of quantitative analysis of calcium ion binding in fusion proteins with PsnPLC, EF chiral domain deletion, and C2 domain deletion. Detailed Implementation

[0018] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0019] Specific Implementation Method 1: This implementation method provides a quantitative analysis method for protein-bound metal ions based on prokaryotic expression:

[0020] I. Prokaryotic expression of the target protein: The ORF sequence (without the stop codon) of the target protein is inserted into a prokaryotic expression vector to construct a recombinant plasmid, which is then transformed into competent cells to obtain a prokaryotic expression strain, which is then induced to express the protein.

[0021] II. Purification of the target protein: Collect the bacterial cells obtained in step one, break them up, and then purify the fusion protein to obtain the purified fusion protein;

[0022] III. Quantitative analysis of protein-bound metal ions: The purified fusion protein was incubated with metal ions, purified again, and the supernatant was collected. The final content of metal ions was detected to indirectly quantify protein-bound metal ions.

[0023] Specific Implementation Method Two: This implementation method is the same as Specific Implementation Method One in that the prokaryotic expression vector is the pGEX-KG vector. Everything else is the same as in Specific Implementation Method One.

[0024] Specific Implementation Method 3: This implementation method differs from Specific Implementation Method 1 or 2 in that: if the fusion protein is located in the supernatant after fragmentation, the supernatant is incubated with the tag protein purification medium and then eluted for purification. Everything else is the same as in Specific Implementation Method 1 or 2.

[0025] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods One to Three in that: if the fusion protein is expressed in the form of inclusion bodies after fragmentation, the inclusion bodies are dissolved using urea or guanidine hydrochloride, then urea or guanidine hydrochloride is removed by dialysis, and the protein is refolded before incubation with the tagged protein purification medium, followed by elution and purification. Everything else is the same as in Specific Implementation Methods One to Three.

[0026] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods One to Four in that the tagged protein purification medium is a GST tagged purification medium. Everything else is the same as in Specific Implementation Methods One to Four.

[0027] Specific Implementation Method Six: This implementation method differs from Specific Implementation Methods One to Five in that it calculates the amount of protein-bound metal ions by comparing the metal ion content before binding with the metal ion content in the supernatant. Otherwise, it is the same as Specific Implementation Methods One to Five.

[0028] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Methods One through Six in that the metal ion content is determined using an enzyme-linked immunosorbent assay (ELISA) reader in conjunction with a metal ion detection kit. Everything else is the same as in Specific Implementation Methods One through Six.

[0029] Specific Implementation Method Eight: This implementation method differs from Specific Implementation Methods One to Seven in that the metal ion is a calcium ion, iron ion, magnesium ion, or copper ion. Everything else is the same as in Specific Implementation Methods One to Seven.

[0030] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Methods One to Eight in that the target protein is the *Populus simonii* PsnPLC protein or a domain-deleted mutant thereof. Everything else is the same as in Specific Implementation Methods One to Eight.

[0031] Specific Implementation Method Ten: This implementation method differs from Specific Implementation Methods One through Nine in that the domain deletion mutant is plc. △aa1-107 or PLC △aa454-587 The rest is the same as in any of the specific embodiments one to nine.

[0032] The beneficial effects of the present invention are verified using the following embodiments:

[0033] Example 1: Prokaryotic expression and purification of fusion protein

[0034] 1. Construction of prokaryotic expression strain for fusion protein

[0035] pGEX-KG-PsnPLC, plc △aa1-107 (EF hand-shaped structural domain missing), plc △aa454-587 (C2 domain missing) Protein structure diagram as shown below Figure 1As shown, pGEX-KG-PsnPLC originates from patent 2020115730642, "PsnPI-PLC, the gene encoding phosphatidylinositol-specific phospholipase C in Populus tomentosa, and its application." △aa1-107 The amino acid sequence is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 2; plc △aa454-587 The amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4.

[0036] PLC was obtained by PCR using the following primers. △aa1-107 cDNA fragment:

[0037] TrunEF-F-Sma I: 5′-TCCCCCGGG TATGACAGCCTCCATT-3′,

[0038] R-Sac I: 5′-CGAGCTCCAACA AATGGACTTCACA-3′.

[0039] PLC was obtained by PCR using the following primers. △aa454-587 cDNA fragment:

[0040] PsnPLC-F-Sma I: 5′-TCCCCCGGGACGAACAACACT-3′,

[0041] TrunC2: 5′-CGAGCTCTCACTTGGCTTTGGGATCAAATA-3′.

[0042] The PCR system is as follows:

[0043] 2X Taq mix 5 μL

[0044] Primer-F 0.2 μL

[0045] Primer-R 0.2 μL

[0046] 1 μL of *Populus simonii* cDNA template

[0047] Add ddH2O to make up the volume to 10 μL.

[0048] The reaction program was as follows: 94℃ pre-denaturation for 2-3 min; 94℃ denaturation for 30 s, 62℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; 72℃ extension for 2 min. The target fragment was recovered using a gel extraction kit. The purified PCR product and pGEX-KG vector were double-digested with Sma I and Sac I, and ligated using T4 ligase. The ligation system is as follows:

[0049] Ligase 10× Buffer 1 μL

[0050] 100 ng of adhesive recovery carrier

[0051] PCR product 20 ng after enzyme digestion and purification

[0052] T4 DNA Ligase 1 μL

[0053] Add nuclease-free water to bring the volume to 10 μL.

[0054] The ligation system was used to transform *E. coli* BL21(DE3) competent cells, and positive clones were screened by sequencing. Plasmids were extracted and transformed into BL21(DE3) competent cells to construct the prokaryotic expression strain plc. △aa1-107 plc △aa454-587 Store frozen at -80℃ for later use.

[0055] 2. Inducing prokaryotic expression of fusion protein

[0056] The prokaryotic expression method was performed according to the instructions for Weidi Bio BL21(DE3) competent cells to obtain induced bacterial cells.

[0057] 3. Fusion protein purification

[0058] After induction, centrifuge the bacterial cells, remove the supernatant, and resuspend them in 3-4 mL of pre-chilled PBS. Add lysozyme to a final concentration of 1 mg / mL, gently agitate, and incubate on ice for 10-30 minutes. Add Triton (final volume ratio 0.2%), mix vigorously, and incubate on ice for 10 minutes. Transfer the system to sonication tubes, sonicate to disrupt the cell structure, centrifuge, collect the supernatant, and transfer it to new tubes for cryopreservation.

[0059] The supernatant was incubated with GST-tagged protein purification medium and then eluted for purification. The purification and elution process was carried out in accordance with the Beyotime GST-tagged protein purification medium instructions to obtain the purified fusion protein.

[0060] Example 2: Quantitative analysis of calcium ions bound to fusion protein

[0061] 1. Analysis of calcium ion binding and gel migration rate

[0062] Conduct calcium ion binding experiments according to the following reaction system.

[0063] ddH2O 50 μL

[0064] 30 μL of purified fusion protein

[0065] Tris-Cl (pH 7.4) 20 μL

[0066] CaCl2 (10 mM) 100 μL

[0067] Incubate at 20-25℃ for 30 min-1 h, then add 200 μL Tris-Cl to terminate the reaction. Gel migration rate analysis was performed using SDS-PAGE. Protein binding efficiency is shown in the figure. Figure 2-4 In each figure, lane 1 represents the purified fusion protein. In lane 2, the purified fusion protein showed a decreased gel migration rate after incubation with calcium ions, while in lane 3, the purified fusion protein's migration rate returned to normal after the addition of calcium ions / EGTA. These results indicate that the purified fusion proteins can bind to calcium ions. Subsequently, 100 μL of GST-resin was added, and the mixture was incubated on ice for 5–8 h. After centrifugation, 10 μL of the supernatant was used for subsequent calcium ion detection (treatment without the target protein served as a control).

[0068] 2. Quantitative analysis of calcium ions bound to fusion proteins

[0069] The calcium ion content was determined according to the Beyotime calcium content assay kit instructions, using GST empty-tagged protein as a control. The experimental results are as follows: Figure 5 As shown in Table 1:

[0070] Table 1. Results of calcium content determination

[0071]

[0072] The results showed that the recombinant fusion proteins PsnPLC and plc △aa1-107 (EF hand-shaped structural domain missing), plc △aa454-587 All three domains can bind calcium ions in vitro. There was no significant difference in calcium ion binding at pH 6.8, but pH 8.8 significantly increased the calcium ion binding rate. Furthermore, the binding efficiency was significantly higher after the C2 domain was removed than that after the EF chiral domain was removed compared to PsnPLC and PsnPLC. These results indicate that the EF chiral domain may be the main calcium ion binding region in *Populus simonii* PLC, while the removal of the C2 domain increases the calcium ion binding efficiency of the EF chiral domain. The two domains may compete for calcium ion binding, and selective binding to calcium ions may affect the biological function of *Populus simonii* PLC.

[0073] In summary, this method enables in vitro quantitative analysis of protein-bound metal ions through prokaryotic expression.

[0074] For any procedures not specifically described in the above examples, please refer to "Molecular Cloning: A Laboratory Manual" or follow the kit instructions.

Claims

1. A method for quantitative analysis of a protein binding metal ion based on prokaryotic expression, characterized by, The method is: I. Prokaryotic expression of the target protein: the ORF sequence of the target protein is inserted into a prokaryotic expression vector to construct a recombinant plasmid, which is then transformed into competent cells to construct a prokaryotic expression strain, and the target protein is induced and expressed; II. Purification of the target protein: the bacterial cells obtained in step I are collected and broken, and then the fusion protein is purified to obtain the purified fusion protein; III. Quantitative analysis of the protein-bound metal ions: the purified fusion protein is incubated with metal ions, and after being purified again, the supernatant is collected and the final content of the metal ions is detected to indirectly quantify the protein-bound metal ions.

2. The method according to claim 1, wherein the protein is expressed in a prokaryotic cell. The prokaryotic expression vector is a pGEX-KG vector.

3. The method according to claim 1, wherein the protein binding metal ion is quantitatively analyzed based on prokaryotic expression. If the fusion protein is in the supernatant after being broken, the supernatant is incubated with a tag protein purification medium and then eluted and purified.

4. The method according to claim 1, wherein the protein binding metal ion is quantitatively analyzed based on prokaryotic expression. If the fusion protein is expressed in the form of inclusion bodies after being broken, the inclusion bodies are dissolved using urea or guanidine hydrochloride, and then the urea or guanidine hydrochloride is removed by dialysis and the protein is refolded, after which the protein is incubated with a tag protein purification medium and then eluted and purified.

5. The method according to claim 3 or 4, wherein the protein is expressed in a prokaryotic cell. The tag protein purification medium is a GST tag purification medium.

6. The method according to claim 1, wherein the protein is expressed in a prokaryotic cell. The amount of protein-bound metal ions is calculated by comparing the content of metal ions before binding with the content of metal ions in the supernatant.

7. The method according to claim 1, wherein the protein is expressed in a prokaryotic cell. The content of metal ions is determined using an enzyme label instrument in combination with a metal ion detection kit.

8. The method according to claim 1, wherein the protein is expressed in a prokaryotic cell. The metal ions are calcium ions, iron ions, magnesium ions or copper ions.

9. The method according to claim 1, wherein the protein is expressed in a prokaryotic cell. The target protein is a PsnPLC protein of Populus simonii x Populus nigra or a domain deletion mutant thereof.

10. The method according to claim 1, wherein the protein is expressed in a prokaryotic cell. The domain deletion mutant is plc △aa1-107 or plc △aa454-587 .

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