CN115947823B - An expression protein for screening toxic metals that bind and/or activate EGFR and a preparation method thereof - Google Patents

Abstract

The application relates to the field of biotechnology, in particular to the field of biotechnology, and in particular relates to an expression protein for screening toxic metals which bind and/or activate EGFR and a preparation method thereof; the nucleotide sequence of the cDNA for expressing the protein is shown as SEQ ID NO. 1; the method comprises the following steps: introducing the vector into a host cell to obtain a recombinant cell; culturing the recombinant thalli and inducing protein expression, extracting and purifying, and performing enzyme digestion to obtain an expressed protein; selecting a plurality of polypeptide peptide segments which are close to the functional region and free from cysteine residues in the nonfunctional region of EGFR, thereby obtaining cDNA fragments of the expression protein, combining toxic metals by utilizing the cysteine residues in the expression protein, and screening the toxic metals by utilizing the combination characteristic, so that the toxic metals combined and/or activated with EGFR are screened out on the premise of not influencing the normal physiological function of EGFR.

Description

An expression protein for screening toxic metals that bind and/or activate EGFR and a preparation method thereof

Technical Field

The present application relates to the field of biotechnology, and in particular to an expression protein for screening toxic metals that bind to and/or activate EGFR and a preparation method thereof.

Background technique

When humans are exposed to toxic metals, they will induce toxicity and carcinogenicity in the human body, and many important cancer-related pathways will be activated, such as HIF-1α, NF-κB, RAS, and PI3K-Akt. It is reported that long-term exposure to arsenic is associated with lung cancer, liver cancer, bladder cancer, kidney cancer, skin cancer and other cancers; cadmium is classified as a human lung cancer carcinogen, and also plays an important role in prostate cancer, kidney cancer, liver cancer, bladder cancer and gastric cancer; chronic lead exposure can cause lymphoma. Current research on toxic metals focuses on the role of cell surface cysteine residues in heavy metal binding, but due to the binding of cell surface protein receptors and toxic metals, current research is relatively limited.

Epidermal Growth Factor Receptor (EGFR) is an extracellular protein receptor of the epidermal growth factor (EGF) family, which is widely distributed on the surface of various mammalian cells such as epithelial cells, fibroblasts, glial cells, keratinocytes, etc. Existing studies have shown that the process of ligand binding and phosphorylation activation of EGFR is often the initiating event in cell malignant transformation, so the ligand/EGFR pathway is also considered to be the center of cancer research. At present, the research related to toxic metals and cysteine residues on EGFR tends to use the influence of toxic metals on the functional area of EGFR to screen toxic metals that bind or activate EGFR, but ignores the influence of toxic metals on the normal physiological function of EGFR. Therefore, how to provide an expression protein for screening toxic metals that bind and/or activate EGFR, so as to achieve the screening of toxic metals that bind and/or activate EGFR without affecting the normal physiological function of EGFR, is a technical problem that needs to be solved urgently.

Summary of the invention

The present application provides an expression protein and a preparation method for screening toxic metals that bind to and/or activate EGFR, so as to solve the technical problem in the prior art that it is difficult to screen out toxic metals that bind to and/or activate EGFR without affecting the normal physiological function of EGFR.

In a first aspect, the present application provides an expression protein for screening toxic metals that bind to and/or activate EGFR, wherein the nucleotide sequence of the cDNA of the expression protein is shown in SEQ ID NO.1.

Optionally, the cDNA expressing the protein consists of a gene sequence of a polypeptide peptide segment, and the polypeptide peptide segment is located in a non-functional region of EGFR.

Optionally, the polypeptide peptide segment includes a plurality of free cysteine residues close to the functional region of EGFR.

Optionally, the amino acid sequence of the expressed protein is shown in SEQ ID NO.2.

In a second aspect, the present application further provides a vector, wherein the vector comprises a nucleotide sequence of a cDNA, wherein the cDNA is the cDNA of the expression protein described in the first aspect.

In a third aspect, the present application further provides a host cell, wherein the host cell comprises the vector described in the second aspect.

In a fourth aspect, the present application further provides a method for preparing the expressed protein according to the first aspect, the method comprising:

Introducing the vector described in the second aspect into a host bacterial cell to obtain a recombinant bacterial cell;

The recombinant bacteria are cultured and protein expression is induced, followed by extraction and purification to obtain the expressed protein.

Optionally, the introduction method includes at least one of heat shock method, electroporation method and chemical transformation method.

Optionally, the culturing of the recombinant bacteria and inducing protein expression, followed by extraction and purification, and enzyme digestion to obtain the expressed protein specifically includes:

Cultivating the recombinant bacteria until the vector transformation is complete, and then adding an inducer for induction to obtain a strain capable of expressing the tag fusion protein;

The strain is lysed and extracted, and then purified to obtain the expressed protein.

Optionally, the strain is lysed and extracted, and then purified and digested by enzymes to obtain the expressed protein, specifically comprising:

The strain is lysed, and then the tissue is broken and centrifuged to obtain the tag fusion protein;

The tag fusion protein is adsorbed on a column, eluted, separated, purified and digested by enzymes to obtain the expressed protein.

The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

The embodiments of the present application provide an expression protein for screening toxic metals that bind to and/or activate EGFR. The cDNA sequence is designed according to the characteristic polypeptide region of the EGFR protein, and a plurality of polypeptide peptides containing free cysteine residues close to the functional region of the EGFR are selected in the non-functional region of the EGFR. A comprehensive design is performed based on the gene sequences of the selected polypeptide peptides, and the introns in these gene sequences are removed to obtain the cDNA of the expression protein. The designed cDNA sequence can transform and express an expression protein containing a plurality of cysteine residues. The cysteine residues in the expression protein are used to bind to heavy metals, so that the expression protein can bind to toxic metals. This binding principle is used to screen out toxic metals that bind to and/or activate EGFR without affecting the normal physiological function of EGFR.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

FIG1 is a schematic diagram of a preparation method according to an embodiment of the present invention;

FIG2 is a schematic diagram of the expression vector pGEX6P-1-E2 constructed in the examples of the present application;

FIG3 is a diagram showing the detection of a target group after purification of the E2 gene expression tag fusion protein provided in an embodiment of the present application;

FIG4 is a schematic diagram of obtaining the target protein after cleavage of the tag fusion protein;

FIG5 is a schematic diagram of the sequencing results of peptide segments of a partial expression protein E2 provided in the examples of the present application;

FIG6 is a schematic diagram of the sequencing results of peptide segments of a partial expression protein E2 provided in the examples of the present application;

FIG. 7 is a schematic diagram of the sequencing results of peptide segments of a partial expression protein E2 provided in the examples of the present application;

FIG8 is a data diagram showing the result of the toxic metal Hg in the form of HgCl ₂ interacting with the protein E2 provided in the examples of the present application;

FIG9 is a data diagram showing the result of the toxic metal Pd in the form of PdCl ₂ interacting with protein E2 provided in the examples of the present application;

FIG10 is a data diagram showing the result of the toxic metal Cd in the form of CdCl ₂ interacting with the protein E2 provided in the examples of the present application;

FIG11 is a WB result diagram of phosphorylated EGFR after HaCaT cells were exposed to toxic metals provided in the examples of the present application;

FIG. 12 is a WB analysis result diagram of phosphorylated EGFR after HaCaT cells were exposed to toxic metals provided in an example of the present application.

Detailed ways

In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

The creative thinking of this application is:

EGFR is a transmembrane glycoprotein and a tyrosine kinase receptor. Its N-terminus is located outside the cell and is a cysteine (Cysteine, Cys)-rich domain that can bind to ligands such as EGF and TGFα (Transforming Growth Factorα, TGFα), leading to receptor dimerization and activating the tyrosine kinase activity of EGFR. When the tyrosine residue undergoes further conformational changes, EGFR acts as an anchor point for a variety of downstream signaling proteins, thereby initiating related signal transduction, leading to cell apoptosis, proliferation, invasion and metastasis.

Existing studies have made it clear that cysteine residues in proteins can bind to some metals, among which the sulfhydryl site (–SH) has a particularly high affinity for sulfhydryl metals (such as mercury, arsenic, cadmium, copper and lead). When metals occupy the metal binding site on the zinc ion, they coordinate with the cysteine residues to form R-S bonds, which are the key to the interaction with the sulfhydryl groups on the cysteine residues. At the same time, most carcinogenic metals (such as cadmium and lead) are soft acids, while cysteine residues are alkaline. Soft acids and soft bases can form stable complexes through coordination, and the stability constant of the metal-sulfhydryl group formed by sulfhydryl metals (such as mercury, arsenic, and cadmium) among carcinogenic metals is usually several orders of magnitude higher than that of the corresponding metal-carboxyl and metal-phosphoryl complexes.

Therefore, it is reasonable to speculate that these carcinogenic metals can act as ligands to bind to the cysteine residues on EGFR, leading to EGFR phosphorylation, thereby initiating the ligand/EGFR carcinogenic pathway, and therefore these carcinogenic metals are called toxic metals. By utilizing the characteristics of EGFR and toxic metal binding, toxic metals can be screened in turn, so that toxic metals can be screened using expressed proteins.

The present application example provides an expression protein for screening toxic metals that bind to and/or activate EGFR, and the nucleotide sequence of the cDNA of the expression protein is shown in SEQ ID NO.1.

In some optional embodiments, the cDNA of the expressed protein consists of a gene sequence of a polypeptide peptide segment, and the polypeptide peptide segment is located in a non-functional region of EGFR.

In the embodiments of the present application, controlling the specific source of cDNA can ensure that the expressed protein after cDNA conversion and expression is derived from the non-functional region of EGFR, thereby enabling the screening of proteins that bind to and/or activate EGFR when EGFR functions normally.

In some optional embodiments, the polypeptide peptide segment includes a plurality of free cysteine residues close to the functional region of EGFR.

In the embodiments of the present application, controlling the source of the polypeptide peptide segments of the cDNA can ensure that the expressed protein after the cDNA is transformed and expressed has sufficient cysteine residues, thereby ensuring the binding of the expressed protein with toxic metals, thereby enabling the screening of proteins that bind to and/or activate EGFR.

In some optional embodiments, the amino acid sequence of the expressed protein is shown in SEQ ID NO.2.

In the embodiments of the present application, the specific amino acid sequence of the expressed protein is controlled. Since the cDNA of the expressed protein is composed of a gene sequence corresponding to a polypeptide peptide segment of multiple free cysteine residues, and when it is transformed into a host cell for expression, multiple different amino acids will be produced. Therefore, the specific sequence of the amino acid sequence of the expressed protein is controlled, indicating that the designed expressed protein is the closest to the actual EGFR protein amino acid structure.

As shown in FIG. 2 , based on a general inventive concept, an embodiment of the present application further provides a vector, wherein the vector comprises a nucleotide sequence of a cDNA, and the cDNA is the cDNA for expressing the protein described in the first aspect.

The vector realizes its function based on the above-mentioned cDNA expressing the protein. The specific functional nucleotide composition of the vector can refer to the above-mentioned embodiment. Since the vector includes part or all of the technical solutions of the above-mentioned embodiments, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be described one by one here.

Based on a general inventive concept, an embodiment of the present application further provides a host cell, wherein the host cell comprises the vector.

The host cell realizes its function based on the above-mentioned vector. The specific composition of the host cell can refer to the above-mentioned embodiment. Since the host cell includes part or all of the technical solutions of the above-mentioned embodiments, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be described one by one here.

As shown in FIG1 , based on a general inventive concept, the present embodiment further provides a method for preparing an expressed protein, the method comprising:

S1. Introducing the vector into the host cell to obtain a recombinant cell;

S2. Cultivate the recombinant bacteria and induce protein expression, then extract and purify, and perform enzyme digestion to obtain the expressed protein.

In the embodiments of the present application, by introducing a vector into a host bacterial cell and then expressing the introduced vector, an expression protein containing the expected amino acid sequence can be obtained, thereby ensuring the accurate expression of the expression protein.

The preparation method is implemented based on the above-mentioned carrier. The specific carrier involved in the preparation method can refer to the above-mentioned embodiment. Since the preparation method includes part or all of the technical solutions of the above-mentioned embodiments, it at least has all the beneficial effects brought by the technical solutions of the above-mentioned embodiments, which will not be repeated here one by one.

In some optional embodiments, the introduction method includes at least one of heat shock method, electroporation method and chemical transformation method.

In the embodiments of the present application, the specific method of introducing the vector into the host cell is controlled to ensure that the vector can be introduced into the host cell in a variety of ways. At the same time, in order to facilitate the operation, the heat shock method is preferred.

In some optional embodiments, the culturing of the recombinant bacteria and inducing protein expression, followed by extraction and purification to obtain the expressed protein, specifically includes:

S201. Cultivating the recombinant bacteria until the vector transformation is complete, and then adding an inducer for induction to obtain a strain capable of expressing the tag fusion protein;

S202. Lyse and extract the strain, and then purify and digest it with enzymes to obtain the expressed protein.

In the embodiments of the present application, the specific process of controlling the expression of the expressed protein by the recombinant bacteria can ensure that a relatively accurate expressed protein is obtained, thereby ensuring the sufficiency of cysteine residues in the expressed protein, ensuring the binding properties of EGFR and toxic metals, and paving the way for toxic metals that bind to and/or activate EGFR.

In some optional embodiments, the strain is lysed and extracted, and then purified to obtain the expressed protein, specifically comprising:

S212. Lysing the strain, then breaking the tissue and centrifuging to obtain a tagged fusion protein;

S222. The tag fusion protein is adsorbed on a column, then eluted, separated, purified and digested by enzymes to obtain the expressed protein.

In the embodiments of the present application, the specific steps of extracting the expressed protein from the strain are controlled, and the tag fusion protein can be used to screen the appropriate expressed protein, and then the tag fusion protein is separated and purified, thereby removing the tag to obtain a pure expressed protein.

The present application will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are intended only to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.

Example 1

1. Construction of the expression vector pGEX6P-1-E2 for the structural peptide fragment of E2 protein

Specific steps are as follows:

(1) Synthesize a cDNA containing the structural peptide of the E2 protein as shown in SEQ ID NO: 1, wherein the specific sequence is: 5′-GACACCTGCCCCCCACTCATGCTCTACAACCCCACCACGTACCAGATGGATGTGAACCCCGAGGGCAAATACAGCTTTGGTGCCACCTGCGTGAAGAAGTGTCCCCGTAATTATGTGGTGACAGATCACGGCTCGTGCGTCCGAGCCTGTGGGGCCGACAGCTATGAGATGGAGGAAGACGGCGTCCGCAAGTGTAAGAAGTGCGAAGGGCCTTGCCGCAAAGTGTGTA ACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCTACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCTGA-3', then the T/A clone of the synthesized cDNA sequence was double-digested with EcoRI/XhoI, the target band was recovered, and connected with the expression vector plasmid pGEX6P-1 (as shown in Figure 2) double-digested with EcoRI/XhoI to obtain a connection product; wherein, pGEX6P-1 (resistance is Amp, the label is GST) is modified on the basis of the VECTOR Escherichia coli protein expression vector commonly used internationally, and is a protein expression vector carrying the LacI gene, lacUV5 promoter and T7 RNA polymerase gene.

The pGEX6P-1 vector in this application was purchased from Transduction Advanced Biotechnology Co., Ltd. (Wuhan).

The endonucleases EcoRI/XhoI and T4 ligase used for ligation were both from Transduction Biotechnology (Wuhan) Co., Ltd., and the usage and dosage were in accordance with the product instructions provided by the company.

(2) The ligation product was introduced into Escherichia coli DH5α (purchased from Shanghai Angyu Biotechnology Co., Ltd.) by heat shock method and cultured on LA resistant medium containing 250 ppm ampicillin (purchased from Roche Biotechnology Co., Ltd.). The formula of LA is shown in Molecular Cloning Experiment Guide (3rd edition), written by J. Sambrook, EF Fritsch, T. Maniatis, translated by Huang Peitang, Wang Jiaxi, etc., Science Press, 2002 edition.

(3) The single colony grown on the LA resistance medium was inoculated into a sterilized 10 mL centrifuge tube on a clean bench. 3 mL of LB resistance medium containing 250 ppm kanamycin was added to the tube in advance, and then cultured on a shaker at 37°C for 16 h to 18 h. The plasmid was then extracted according to the method described in Molecular Cloning Laboratory Manual (J. Sambrook and D.W. Russell, translated by Huang Peitang et al., Science Press, 2002), and then EcoRI/XhoI double enzyme digestion and PCR detection were performed. The positive expression plasmid vector was obtained according to the size of the inserted fragment and named pGEX6P-1-E2.

(4) The expression vector pGEX6P-1-E2 of step (3) was introduced into the Rosetta (DE3) (purchased from Shanghai Ang Yu Biotechnology Co., Ltd.) strain by the heat shock method as shown in step (2), and the transformed strain was named T-E2.

Example 2

Comparing Example 2 with Example 1, the difference between Example 2 and Example 1 is:

2. Induced expression and purification of soluble GST-tagged fusion proteins in E. coli

IPTG (Isopropyl-beta-D-thiogalactopyranoside) was added to the T-E2 strain to induce the expression of a GST-tagged fusion protein, which was then extracted, purified, and finally digested to obtain the target protein without a GST tag. The kit used was a GST-tagged protein purification kit (purchased from Beyotime). The specific steps are as follows:

1. GST-tagged protein induced expression

(1) Take 50 μL of the seed culture solution at -80°C, add it to 5 mL of LB resistance medium containing 250 ppm kanamycin, then culture it on a shaker at 37°C for 16 h to 18 h, then add it to 300 mL of LB resistance medium containing 250 ppm kanamycin, and then culture it on a shaker at 37°C for 4 h to 6 h until the logarithmic growth phase.

(2) Add IPTG at a final concentration of 0.5 mM and induce expression at 4°C and 140 rpm for 32 h.

2. Extraction of total protein

(1) Take out the bacterial solution and centrifuge it at 6000g for 10 min at 4°C. Discard the supernatant, weigh the bacteria, add 4 mL of lysis buffer per gram of wet weight of bacterial pellet, fully resuspend, add protease inhibitors and lysozyme, and place on ice to continue to fully lyse for 30 min.

(2) Take out the bacterial lysate and disrupt it in a low-temperature ultrasonic tissue disruptor.

(3) Add RNase at a final concentration of 10 μg/mL and DNase I at 5 μg/mL and continue lysis on ice for 10 min.

(4) Centrifuge at 12,000 g for 20 min at 4°C. Collect the supernatant and place on ice. Take out 50 μL of the supernatant and record it as CL for subsequent detection.

3. Purification of GST-tagged Protein

(1) Adsorption and column attachment of GST-tagged proteins:

1) Take 1 mL of BeyoGold GST-tag resin that has been shaken evenly and place it in a 1.5 mL centrifuge tube. Centrifuge at 4°C (condition: 1000 g × 20 s). Aspirate the supernatant and add 0.5 mL of lysis buffer to the resin to fully resuspend it. Repeat the balancing process twice and aspirate the supernatant to obtain dry resin:

2) Add 4 mL of bacterial lysate supernatant to the equilibrated resin and slowly mix and adsorb on a horizontal shaker at 4°C for 2 h to 3 h.

(2) Washing, elution and separation and purification of GST protein on the column:

1) Add the sample that has been fully loaded onto the column into the empty column tube of the affinity chromatography column provided in the kit using a pipette tip with a cut tip;

2) Wait for a while until there is obvious solid-liquid separation, open the bottom cover, and collect the flow-through liquid, which is recorded as FT;

3) After the flow-through has run out, add 0.8 mL of lysis buffer (containing 0.2% Tween-20) to wash the column protein. Repeat the washing process for 15 times. Collect the wash effluent and record it as Wn (n=1, 2…15).

4) Use 0.4 mL elution buffer (containing 10 mM 1×GSH) for elution, repeat the elution of the target tagged protein 5 times, collect the eluate, record it as Eln (n=1,2…5), and store each protein sample at -80°C.

(3) Protein gel testing

50 μL of CL, FT, W1, W15, and El1-5 protein samples were taken respectively, added with 12.5 μL of 5× Loading Buffer (containing 5% beta-mercaptoethanol), mixed, and heated in a metal bath at 100°C for 5 min for denaturation; then, 15% SDS-PAGE was run at 100 V for separation, and R250 Coomassie Brilliant Blue was used for protein staining to observe the expression and purification status. The results are shown in Figure 3.

Example 3

Comparing Example 3 with Example 1, the difference between Example 3 and Example 1 is:

3. Enzymatic cleavage of soluble GST-tagged fusion protein and separation and purification of target peptide

Specific steps are as follows:

1. Desalination

(1) Take two elution samples, E12 and E13, mix them evenly, take 50 μL and record it as "before desalting", and cook the sample for subsequent detection;

(2) Take a protein desalting column for pretreatment, centrifuge at 1000g for 2 min to remove the storage solution, add 2.5 mL of enzyme digestion buffer (containing 0.2% Tween-20 and 1 mM DTT) to the column and centrifuge, repeat balancing twice, and discard the buffer in the collection tube;

(3) Add the mixture of E12 and E13 to a centrifugal column at 1000 g for 2 min, collect the filtrate, take 50 μL of the filtrate and mark it as “after desalting”, and cook the sample for subsequent testing;

(4) The protein concentration of the desalted sample was detected by Bradford method to obtain the total amount of GST-tagged fusion protein in the desalted solution.

2. Enzyme Digestion

(1) According to the total amount of GST-tagged fusion protein contained in the desalted solution, 0.04 μL of ppase (PreScission Protease) was added to 1 μg of the desalted sample and digested overnight at 4°C.

(2) Take 50 μL of enzyme digestion solution and add 12.5 μL of 5× Loading Buffer to cook the sample for subsequent detection.

(3) Add 4.4 μl of BeyoGold GST-tag to 1 μg of desalted sample, and take a proportion of BeyoGold GST-tag and pipette it evenly. Centrifuge it in a 1.5 mL centrifuge tube at 4°C and 1000 g for 20 s. Aspirate the supernatant with a pipette with a tip removed, add enzyme cleavage buffer to the resin to fully resuspend it, and repeat the balancing twice.

(4) Add the enzyme cleavage solution to the resin gel and allow to bind at room temperature for 20 to 30 minutes.

(5) Centrifuge at 500 g for 5 min, collect the supernatant, run 15% SDS-PAGE gel for separation, use silver staining kit (Biyuntian) for protein staining, and observe the enzyme cleavage. The results are shown in Figure 4.

As shown in Figure 4, the target band without GST tag was finally obtained.

The ppase (PreScission Protease) reagent used in the above reaction was purchased from Changzhou Tiandirenhe Biotechnology Co., Ltd., IPTG (Isopropyl-beta-D-thiogalactopyranoside) was purchased from Beijing Coolaibo Technology Co., Ltd., and all other reagents were purchased from Biyuntian Company.

Example 4

Comparing Example 4 with Example 1, the difference between Example 4 and Example 1 is:

4. Structural Sequencing of Target Peptide

The obtained target polypeptide was subjected to SDS-PAGE electrophoresis, and after gel cutting and recovery, in-gel enzymatic treatment was performed, and the synthesized target polypeptide was detected on a MICROLC-QTOF-MS machine to obtain its amino acid sequence as shown in SEQ ID NO.2: KDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISG, and the specific steps are as follows:

1. Gel cutting: Place the gel after SDS-PAGE electrophoresis in a glass dish, cut the band, then cut the gel band into small pieces and rinse with distilled water twice.

2. Add an appropriate amount of decolorizing solution and place in a 37°C constant temperature incubator (water bath decolorization) until it is colorless or very light in color.

3. Aspirate the decolorizing solution, add an appropriate amount of acetonitrile to rinse, aspirate the acetonitrile, add acetonitrile again, let it stand for 10 minutes, aspirate the acetonitrile, and use a vacuum centrifugal concentrator to dehydrate and dry the gel block.

4. Add 25 mM dithiothreitol solution (DTT) to the dried gel pieces, place at 55°C, and incubate for 45 min.

5. Add appropriate amount of acetonitrile to rinse, then absorb dryness, add acetonitrile again for 10 minutes, absorb the acetonitrile, and use a vacuum centrifugal concentrator to dehydrate and dry the gel block.

6. Add an appropriate amount of 55 mM iodoacetamide solution and react for 30 minutes in the dark.

7. Rinse with acetonitrile, then soak in acetonitrile for 10 minutes, and use a vacuum centrifugal concentrator to dehydrate and dry the gel block.

8. Add appropriate amount of trypsin to cover the micelles, place upside down in a 37℃ incubator, and incubate for 12h to 16h.

9. Terminate the enzymatic reaction with 1 μL of 10% trifluoroacetic acid solution, centrifuge at low speed to separate the particles and the solution, and collect the solution into a new 2 mL EP tube.

10. Add an appropriate amount of extraction solution (50% acetonitrile, 0.1% formic acid) to the micelles, place at 37°C, incubate for 30 minutes, take the supernatant and combine it with the solution in the EP tube in step 9, and repeat the extraction step twice.

11. Concentrate the supernatant in a vacuum centrifugal concentrator. When about 10 μL remains, take out the centrifuge tube and store it at -20°C.

12. Before loading, add 20 μL of 0.1% formic acid solution to re-dissolve. Vortex to mix and centrifuge, repeat twice (the final solution volume is about 50 μL to 70 μL).

13. Centrifuge at high speed for 5 minutes, take 50 μL of peptide solution and add it to the mass spectrometry loading bottle. Be careful not to have bubbles at the bottom of the bottle and store it at 4°C for mass spectrometry analysis.

The sequencing results are shown in Figures 5 to 7. As shown in Figures 5 to 7, the peptide coverage is 94.4%, proving that the protein purified and digested is indeed the target protein E2. The chromatographic conditions are:

Liquid phase model: MicroLC M5 (SCIEX, USA): C18 chromatographic column (3.0 μm, 0.3 mm×150 mm, Phenomenex, USA), column temperature was 45°C.

Mobile phase A: 2% ACN (containing 0.1% FA, v/v); Mobile phase B: 98% ACN (containing 0.1% FA, v/v). Flow rate: 7 μL/min; Injection volume: 5 μL.

The gradient was set as follows: 5% B (0-2 min), 5%-40% B (2 min-25 min), 40%-85% B (25 min-28 min), 85% B (28 min-33 min), 85%-5% B (33 min-35 min), 5% B (35 min-45 min).

Mass spectrometry conditions: Mass spectrometry model: TripleTOF 5600+ (SCIEX, USA):

Positive ion mode; ion source temperature 350℃; spray voltage 5500V; ion source gas GS1 is 16psi; ion source gas GS2 is 17psi; curtain gas CUR is 30psi; primary mass spectrometry (MS) scanning range 300Da～1250Da; secondary mass spectrometry (MS/MS) scanning range 100Da～1500Da.

Example 5

Comparing Example 5 with Example 1, the difference between Example 5 and Example 1 is:

5. Screening application of target peptides for toxic metals that can bind to EGFR

After E2 was exposed to representative toxic metals Hg, Cd, and Pb in the form of HgCl ₂ , CdCl ₂ , and PbCl ₂ , respectively, the fluorescence intensity at 350 nm was used to represent the tertiary structure state of the target polypeptide under different treatments. The specific steps are as follows:

1. Prepare 20 mM phosphate buffer with a pH of 7.4 and containing 1 mM TCEP; the phosphate buffer is prepared by mixing sodium dihydrogen phosphate and disodium hydrogen phosphate solutions and adjusting the pH to 7.4 using a pH meter to obtain a mixture of the two as a buffer stock solution.

2. Take the E2 protein solution and use a desalting column (purchased from Changzhou Tiandirenhe Biotechnology Co., Ltd.) to exchange its buffer into the above-mentioned phosphate buffer;

3. Prepare HgCl ₂ , CdCl ₂ , and PbCl ₂ solutions using phosphate buffer of the same pH.

4. Expose E2 to HgCl ₂ , CdCl ₂ , and PbCl ₂ , wherein the final concentration of E2 is 10 μM, the final concentrations of HgCl ₂ and CdCl ₂ are 50/100 μM, and the final concentration of PbCl ₂ is 100/500 μM.

5. All samples were incubated at 37°C, 180 rpm for 30 min to ensure full reaction and equilibrium.

6. Use a fluorescence instrument for detection, with an excitation wavelength of 259 nm and an emission fluorescence spectrum range of 290 nm to 450 nm, and record the fluorescence intensity.

The experimental results are shown in Figures 8 to 10. As the concentration of toxic metals Hg ²⁺ , Cd ²⁺ , and Pb ²⁺ increases, the fluorescence intensity of E2 gradually decreases, indicating that toxic metals Hg ²⁺ , Cd ²⁺ , and Pb ²⁺ can bind to E2, thereby changing its fluorescence intensity. Finally, peptides that can bind to and activate EGFR toxic metals are screened.

Example 6

Comparing Example 6 with Example 1, the difference between Example 6 and Example 1 is:

VI. Screening for the functional application of toxic metals on EGFR phosphorylation activation

HaCaT cells were exposed to toxic metals Hg, Cd, and Pb in the form of HgCl ₂ , CdCl ₂ , and PbCl ₂ , respectively. The specific steps are as follows:

1. Cell plating: HaCaT cells were plated in 6-well plates at a density of 3×10 ⁵ cells/well and cultured with DMEM complete medium (containing 1% P/S and 10% FBS) for 36 h.

2. Serum starvation cells: Wash once with PBS, and switch to serum-free DMEM (containing 1% P/S) for serum starvation for 12 hours.

3. Exposure: Treat with HgCl ₂ at final concentrations of 0/0.05/0.1 μM, CdCl ₂ at final concentrations of 0/10/25 μM, and PbCl ₂ at final concentrations of 0/10/25 μM for 30 min.

4. Western Blot Analysis:

1) Sample preparation:

After washing the cells twice with pre-cooled PBS, add 150 μL/well RIPA lysis buffer, continue lysis on ice at 140 rpm for 30 min, collect the cells into a 1.5 mL EP tube, add 37.5 μL 5x loading buffer (containing 5% beta-mercaptoethanol), mix, and cook the sample at 100°C for 10 min;

2) 10% SDS-PAGE electrophoresis;

3) Transfer membrane;

4) Blocking: Block with TBST containing 5% BSA at room temperature for 2 h;

5) Primary antibody: 2% BSA-TBST containing primary antibody, incubated overnight at 4°C, where the primary antibody is: Anti-pEGFR (phosphor Tyrosine 1068) antibody [EP774Y]-Rabbit-170kD; β-αctin-M-42KD

6) Washing membrane: TBST, 10 min/time, 3 times;

7) Secondary antibody: Incubate in 2% BSA-TBST containing secondary antibody at room temperature for 1 hour; the secondary antibodies are: Rabbit and Mouse;

8) Washing membrane: TBST, 10 min/time, 3 times;

9) Use developer to develop and see the result.

The results are shown in Figures 11 and 12. As the concentrations of toxic metals Hg ²⁺ , Cd ²⁺ , and Pb ²⁺ increase, the phosphorylated EGFR increases, indicating that toxic metals Hg ²⁺ , Cd ²⁺ , and Pb ²⁺ can activate EGFR, phosphorylate EGFR, and thus activate downstream signaling pathways.

Among them, HaCaT cells were purchased from the Type Culture Collection of Wuhan University, DMEM medium, P/S, and FBS were purchased from GIBCO, primary antibodies Anti-pEGFR and β-αctin-M were purchased from abcan, secondary antibodies were purchased from Biosharp, and developing solution was purchased from Thermo Fisher.

In summary, the expressed protein protected in the present application can not only successfully express the target protein E2, but also has a screening effect on toxic metals that bind to and/or activate EGFR.

One or more technical solutions in the embodiments of the present application also have at least the following technical effects or advantages:

(1) The embodiments of the present application provide an expression protein for screening toxic metals that bind to and/or activate EGFR. The cDNA sequence is designed according to the characteristic polypeptide region of the EGFR protein. A plurality of polypeptide peptides containing free cysteine residues close to the functional region of the EGFR are selected in the non-functional region of the EGFR. A comprehensive design is performed based on the gene sequences of the selected polypeptide peptides, and the introns in these gene sequences are removed to obtain the cDNA of the expression protein. The designed cDNA sequence can transform and express an expression protein containing a plurality of cysteine residues. The cysteine residues in the expression protein are used to bind heavy metals, so that the expression protein can bind to toxic metals. This binding principle is used to screen out toxic metals that bind to and/or activate EGFR without affecting the normal physiological function of EGFR.

(2) The vector provided in the examples of the present application is not only easy to introduce into host cells, but also can be stably expressed.

(3) The application provided in the examples of the present application uses representative toxic metals Hg ²⁺ , Cd ²⁺ , and Pb ²⁺ to expose the present protein in the form of HgCl ₂ , CdCl ₂ , and PbCl ₂ , respectively. The fluorescence characterization results show that the present protein can bind to the toxic metals, thereby changing their fluorescence values. At the same time, HaCaT cells are exposed to the toxic metals Hg, Cd, and Pb in the form of HgCl ₂ , CdCl ₂ , and PbCl ₂ , respectively. The WB results show that the toxic metals used can activate EGFR in the cells, thereby phosphorylating EGFR, indicating that the expressed protein of the present application can screen toxic metals that bind to or activate EGFR.

(4) The application provided in the embodiments of the present application can screen for toxic metals that bind to and activate EGFR by using designed expression proteins. This has important guiding significance for the research on toxic metals that can bind to and activate EGFR, and provides a new method for screening toxic metals that bind to and activate EGFR.

Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present application; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

In the present application, in the absence of any contrary description, the directional words used, such as "upper" and "lower", are specifically the directions of the drawings in the accompanying drawings. In addition, in the description of the present specification, the terms "including", "comprising", etc. refer to "including but not limited to". In this article, relational terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. In this article, "and/or" describes the association relationship of the associated objects, indicating that there may be three relationships, for example, A and/or B, which can represent: A exists alone, A and B exist at the same time, and B exists alone. Wherein A, B can be singular or plural. In this article, "at least one" refers to one or more, and "plural" refers to two or more. "At least one", "at least one of the following" or similar expressions refer to any combination of these items, including any combination of singular or plural items. For example, "at least one of a, b, or c" or "at least one of a, b and c" can both mean: a, b, c, a-b (i.e. a and b), a-c, b-c, or a-b-c, where a, b, c can be single or plural, respectively.

The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features applied for herein.

Claims (10)

1. An expression protein for screening toxic metals that bind to and/or activate EGFR, characterized in that the nucleotide sequence of the cDNA of the expression protein is as shown in SEQ ID NO.1. 2. The expression protein according to claim 1 is characterized in that the cDNA of the expression protein is composed of a gene sequence of a polypeptide peptide segment, and the polypeptide peptide segment is located in a non-functional region of EGFR. 3. The expressed protein according to claim 2, characterized in that the polypeptide peptide segment comprises a plurality of free cysteine residues close to the functional region of EGFR. 4. The expressed protein according to claim 1, characterized in that the amino acid sequence of the expressed protein is shown in SEQ ID NO.2. 5. A vector, characterized in that the vector comprises a nucleotide sequence of cDNA, and the cDNA is the cDNA expressing the protein according to any one of claims 1 to 4. 6. A host cell, characterized in that the host cell comprises the vector according to claim 5. 7. A method for preparing the expression protein according to any one of claims 1 to 4, characterized in that the method comprises: Introducing the vector as claimed in claim 5 into a host bacterial cell to obtain a recombinant bacterial cell; The recombinant bacteria are cultured and protein expression is induced, followed by extraction and purification, and enzyme digestion to obtain the expressed protein. 8. The method according to claim 7, characterized in that the introduction method includes at least one of heat shock method, electroporation method and chemical transformation method. 9. The method according to claim 7, characterized in that the culturing of the recombinant bacteria and inducing protein expression, followed by extraction and purification to obtain the expressed protein, specifically comprises: Cultivating the recombinant bacteria until the vector transformation is complete, and then adding an inducer for induction to obtain a strain capable of expressing the tag fusion protein; The strain is lysed and extracted, and then purified and digested with enzymes to obtain the expressed protein. 10. The method according to claim 9, characterized in that the lysis and extraction of the strain and subsequent purification to obtain the expressed protein specifically comprises: The strain is lysed, and then the tissue is broken and centrifuged to obtain the tag fusion protein; The tag fusion protein is adsorbed on a column, eluted, separated, purified and digested by enzymes to obtain the expressed protein.