Method for realizing efficient selenium labeling of protein

Abstract

The invention discloses a method for realizing high-efficiency selenium labeling of protein, which comprises the following steps of: in the process of expressing plasmids containing target protein through a prokaryote expression system, realizing high-efficiency replacement of cysteine sulfur atoms in the target protein by selenium atoms by regulating and controlling the ratio of selenium to sulfur elements in a culture medium; on the premise of keeping a good growth state of cells, the selenium labeling efficiency can reach 86%, and the protein expression quantity is stabilized at about 1mg / g (protein / thallus wet weight); structural analysis shows that the secondary structure of the obtained selenium-labeled protein is basically consistent with that of natural protein and is not obviously changed. The method is easy and convenient to operate, high in labeling efficiency, high in protein yield, good in repeatability and suitable for structural biology research such as nuclear magnetic resonance, an effective means is provided for research of the functional mechanism of sulfur atoms in biomacromolecules (including small peptides, polypeptides, peptide fragments, proteins and protein or peptide complexes) with selenium as a probe, and the method has a wide application prospect. Good scientific research application prospects and industrial popularization values are realized.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for realizing efficient selenium labeling of proteins, namely a method for efficiently replacing sulfur atoms of cysteine ​​in proteins with selenium atoms. Background Art

[0002] Sulfur plays a crucial role in living organisms, particularly cysteine ​​(Cys). As a key component of protein active centers, it is widely present in various organisms and its abundance increases with evolution, indicating its crucial role in regulating higher-order biological structures. Cys's primary functions include promoting proper protein folding, regulating redox states, and participating in the functional regulation of active centers. However, existing protein structure determination methods, such as mass spectrometry, spectroscopy, X-ray diffraction, and nuclear magnetic resonance (NMR), are unable to directly detect sulfur atoms, limiting mechanistic studies of biological processes in which sulfur atoms participate.

[0003] Selenium atoms have similar chemical properties to sulfur atoms and are therefore used as the target of nuclear magnetic resonance (NMR) 77 Se NMR) is an ideal alternative for the detection of sulfur atoms. 77 Se isotope, with a nuclear spin quantum number of 1 / 2 and a natural abundance of 7.6%, is suitable for NMR detection. Studies have shown that selenocysteine ​​(Sec) not only has unique chemical properties, such as a lower pKa value that makes it easier to deprotonate and participate in nucleophilic reactions, but also its larger atomic radius gives it unique reaction characteristics under specific environments. Early studies through X-ray crystal diffraction found that selenium labeling has little effect on protein structure, confirming its feasibility as a non-interference marker. In addition, 77 Se NMR technology has made significant progress in recent years, with a chemical shift range of up to 2000 ppm. It is highly sensitive to the environment and is suitable for precise characterization of complex systems.

[0004] For the study of sulfur-containing proteins, such as cytochrome c (cyt c), its active center is covalently linked to the peptide chain by the heme cofactor through the cysteine ​​side chain, and exhibits different coordination states under oxidative stress conditions. Traditional characterization methods such as mass spectrometry and Raman spectroscopy can reveal the coordination state, but it is difficult to intuitively reflect the dynamic conformational change process. In contrast, NMR technology has shown unique advantages in analyzing the relationship between protein function and structure due to its ability to dynamically monitor structural changes. By introducing selenocysteine ​​into the active center of cyt c, it can be used 77 Se NMR technology can monitor the conformational changes of the active center of cyt c in biological processes in real time, thereby gaining a deeper understanding of the relationship between its structure and function.

[0005] However, to achieve the use of77 Se NMR technology is used to study the structure-activity relationship of sulfur-related proteins. First, it is necessary to prepare selenium-labeled protein samples. The existing methods of selenium-labeled proteins, such as the selenium-sulfur inorganic salt substitution method, the 20 amino acid addition method, and the C321 system amber codon expression method, have low labeling efficiency or unstable yields, and cannot specifically label cysteine ​​sulfur atoms, so they cannot be applied to 77 Therefore, it is urgent to develop an efficient method for preparing selenium-labeled proteins and to distinguish between cysteine ​​and methionine sites at the same time. Summary of the Invention

[0006] The present invention is based on the E. coli expression system. By precisely controlling the supply ratio of sulfur and selenium in the culture medium, it successfully achieves the specific selenium labeling of sulfur atoms in cysteine ​​residues in proteins, providing key technical support for the direct analysis of the molecular mechanism of cysteine ​​in protein conformation and function regulation using nuclear magnetic resonance (NMR) technology. The core of this technology is based on the similarity of the physicochemical properties of selenium atoms and sulfur atoms (atomic radius difference <0.2Å). By establishing a dynamic equilibrium strategy of decreasing sulfur source gradient and increasing selenium-labeled amino acid concentration in the culture medium, the selenium labeling efficiency of the target protein is significantly improved (>85%). Finally, a nuclear magnetic grade selenium-labeled protein sample is obtained through high-density fermentation, combined with 77 Se NMR spectrum analysis can accurately track the structural dynamic changes of cysteine ​​sites.

[0007] This approach minimizes the toxic effects of selenium on host cells (increasing cell viability to 82%) by systematically optimizing multiple parameters, including amino acid metabolic balance, operational feasibility, target protein yield (up to 1 mg / L), and labeling efficiency (86%). The solution is universally applicable to expression systems and plasmid design, making it widely applicable to various cysteine-containing protein systems (including free sulfhydryl groups and proteins involved in sulfur coordination bonds), significantly expanding the compatibility of selenium labeling technology.

[0008] The method for preparing cytochrome c labeled with cysteine ​​proposed by the present invention comprises the following steps:

[0009] The first step is the expression of the target protein labeled with selenium:

[0010] 1. Conversion:

[0011] The target protein plasmid was transferred into E. coli, and the transferred E. coli was evenly spread on LB solid medium and cultured in a 37°C incubator overnight;

[0012] Take a portion of the prepared competent state (stored in a -80℃ refrigerator), add the plasmid to a final concentration of 100-150ng / μL, gently tap the bottom of the test tube to mix the contents evenly, place it on ice for 30 minutes, then place the Eppendorf tube in a 42℃ constant temperature water bath for 90 seconds, place it on ice for 1 minute, add 500μL LB (without antibiotics), and culture it on a shaking table at 37℃ and 220rpm for 30-60 minutes. Centrifuge it, re-dissolve it with 100μL culture medium and spread it on a plate (ampicillin resistance), and invert it to culture in a 37℃ constant temperature incubator overnight.

[0013] 2. Pick an order:

[0014] Prepare the culture medium, which consists of 50mM Na2HPO4, 50mM KH2PO4, 50mM NH4Cl, and 10mM NaCl in a 1L conical shake flask, sterilize it at high temperature, and add MgCl2, trace metal mixture, vitamin mixture, and vitamin B after cooling. 12、 Glucose, calcium chloride, ampicillin and the precursor for the synthesis of heme prosthetic group (Heme).

[0015] 3. Expression:

[0016] Transfer 20 mL of this culture medium to a small Erlenmeyer flask. Pick a single colony from the plate in the previous step and transfer it to the 20 mL flask. Supplement with 5 mM Na₂SO₄ and incubate for approximately 13-15 hours. Measure the OD value to 1-1.5. Transfer 1 mL to a 1 L flask and incubate overnight. Supplement with 50 μM Na₂SO₄. The next day, when the OD value is approximately 0.75, add the first dose of 22.5 μM selenocystine and 5 μM methionine. Maintain at 37°C, 220 rpm for 1 hour. Afterwards, add 1 mM of the inducer and incubate at 30°C, 70 rpm for 6 hours. Supplement with the same proportions of selenocystine and methionine. Continue incubating overnight until the next morning, then harvest the cells by centrifugation at 6000 rpm for 15 minutes.

[0017] Step 2: Purification of cytochrome c labeled with cysteine:

[0018] 1. Remove the bacteria after centrifugation to remove the supernatant or store at -20℃, resuspend in 40mL lysis buffer (50mM Tris-HCl (6.057g / L), 5mM EDTA-2Na (1.86g / L), pH 7.5), add lysozyme (Eggwhite, 1.5mg / g lysozyme / wet bacteria) and 5μL DNA digestion enzyme (DNaseⅠ) and digest for 1h.

[0019] 2. Repeatedly disrupt the cells using high pressure, and then centrifuge at 20,000 rpm for 30 minutes.

[0020] 3. Place the supernatant after centrifugation in a clean 250ml beaker and slowly add ammonium sulfate (150g / L) for salting out for 3-12 hours.

[0021] 4. Centrifuge at 20,000 rpm for 30 minutes, remove the supernatant and dialyze overnight (5 L: Na2HPO4 8.66 g, NaH2PO4 4.68 g, pH 7.0).

[0022] 5. Centrifuge the dialyzed solution overnight at 20,000 rpm for 30 min to remove the precipitate and prepare for column purification.

[0023] 6.SP cationic column purification.

[0024] 7. Purification by molecular sieve pg75 column.

[0025] Step 3: Protein spectrum identification:

[0026] Selenium-labeled proteins were characterized using electrospray ionization mass spectrometry (ESI-MS). An acetonitrile-water mobile phase was used (the target protein was eluted at 50% acetonitrile). Using selenium-labeled cytochrome c as an example, the results showed that 46% of the protein was labeled with single-cysteine ​​selenoproteins, 40.1% with double-cysteine ​​selenoproteins, and 13.7% with no selenoproteins. Secondary mass spectrometry confirmed that the double-selenium labeling site was precisely located at the cysteine ​​residues (Cys14 / 17) in cytochrome c that coordinate the heme iron. The highest single-residue labeling efficiency reached 86%, and the target protein yield was consistently 1 mg / g (protein / wet cells).

[0027] The present invention also provides a cytochrome c selenium nuclear magnetic resonance (S-NMR) of selenium-labeled cysteine ​​prepared by the above method. 77 The application of Se NMR to characterize protein conformation is used to study the functional mechanisms of biological processes related to cysteine ​​sulfur atoms at the molecular and atomic levels.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] 1. Current selenium-labeled protein preparation technology has the following main limitations:

[0030] (1) Chemical modification: Selenium-containing small molecules are coupled to proteins through chemical reactions. Although the operation is simple, it is only applicable to sites containing free sulfhydryl groups. Exogenous modification groups are prone to induce steric hindrance effects (α-helix twisting degree ≥ 15%), interfering with the natural conformation of the protein;

[0031] (2) Solid-phase synthesis: limited by the efficiency of polypeptide chain extension and the difficulty of folding and refolding, it is only applicable to small molecular weight proteins (<10 kDa), and the synthesis cost increases exponentially with the number of amino acids;

[0032] (3) Shortcomings of the four mainstream methods for prokaryotic expression:

[0033] 1) Selenium-sulfur inorganic salt replacement method ( Figure 2 、 Figure 3 :By gradually replacing sulfate (SO4 2- ) is selenite (SeO3 2- ) Selenium labeling was achieved, but mass spectrometry analysis showed that the selenium labeling site was mainly the sulfur atom in methionine, and specific selenium labeling of cysteine ​​could not be performed;

[0034] 2) 20 kinds of amino acids are added separately ( Figure 4 ): 20 kinds of amino acids were added to the culture medium respectively, among which selenocysteine ​​was directly added instead of natural cysteine. Although it can achieve cysteine ​​site-specific labeling, the labeling efficiency is low;

[0035] 3) Modification of methionine labeling method: 13 C-methionine isotope labeling strategy, but mass spectrometry results showed that no labeling was successful;

[0036] 4) C321 amber codon system: This system, based on stop codon reprogramming and unnatural amino acid insertion, requires co-expression of six auxiliary proteins, including SelD and CCHL, and relies on the C321ΔRF1 genetically engineered strain. This system is complex to construct, has demanding expression conditions, and results in low target protein yields.

[0037] 2. The method proposed in the present invention overcomes the above-mentioned problems. Compared with the above methods, the present invention is simple to operate, has a selenium labeling efficiency of up to 86%, and a high and stable protein yield of 1 mg / g protein / wet cells. It can also achieve specific labeling of cysteine ​​sites. The method of the present invention also has excellent compatibility and universality, and can be used to fill the gap in the study of the functional mechanism of sulfur atoms in biological systems, with high scientific research application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the mass spectrometry identification result of cytochrome c labeled with cysteine ​​selenium prepared in Example 1. Cytochrome c labeled with selenium at a single cysteine ​​site (Se-ycc) accounts for 46%, and cytochrome c labeled with selenium at two cysteine ​​sites (Se2-ycc) accounts for 40.1%. Therefore, the proportion of selenium labeled at a single cysteine ​​site reaches 86.1%. ycc represents unlabeled cytochrome c.

[0039] Figure 2The primary mass spectrometry statistical results of selenocytochrome c expressed by the selenium-sulfur inorganic salt replacement method in Example 2 are shown on the left for selenocytochrome c expressed at a selenium-sulfur inorganic salt ratio of 0.5 (1:1), and on the right for selenocytochrome c expressed at a selenium-sulfur inorganic salt ratio of 0.9 (9:1).

[0040] Figure 3 The secondary mass spectrometry statistics of selenium cytochrome c expressed by the selenium-sulfur inorganic salt replacement method in Example 2 showed that the selenium labeling site accounted for 72.83% at methionine 80 and 9.68% at methionine 64.

[0041] Figure 4 The mass spectrometry analysis structure of selenocytochrome c expressed by the addition of 20 amino acids in Example 3. 600 When amino acids are added (OD 600 =1, the right side is OD 600 = 1.5), the efficiency of selenium labeling was even at OD 600 When it is 1.5, it is still less than 50%.

[0042] Figure 5 To confirm that the selenium-labeled protein expressed by the new method in Example 1 has no effect on the protein structure, the oxidized (OX) and reduced (re) samples of the selenium-labeled cytochrome c prepared in Example 1 were compared with the wild-type cytochrome c sample, respectively. The circular dichroism results showed that the structure of the prepared selenium-labeled cytochrome c was almost the same as that of the original wild-type cytochrome c, indicating that the method in Example 1 can be used for the study of biological molecular structures.

[0043] Figure 6 This is the mass spectrum of the selenocysteine ​​ubiquitin protein (se-UB) expressed in Example 4. 8540 Da is the unlabeled molecular weight of the protein, and 8600 Da is the molecular weight of the protein after selenium labeling. After selenium labeling, a methyl group is bound to the corresponding site, resulting in an increase of 60 Da (pure selenium labeling results in an increase of 46 Da).

[0044] Figure 7 The one-dimensional cytochrome c of cytochrome c labeled with cysteine ​​prepared in Example 1 at a concentration of 1.5 mM 77 The Se NMR spectrum, with a sampling time of 9 h 30 min, clearly shows the signal of selenium atoms.

[0045] Figure 8 The two-dimensional structure of cytochrome c of selenocysteine ​​prepared in Example 1 in the oxidized and reduced states 1 H, 77 The Se-HSQC correlation spectrum shows that the selenium atoms introduced into the protein by the present invention can serve as a probe to react to changes in protein conformation. DETAILED DESCRIPTION

[0046] In order to make the technical means, creative features, workflows, and methods of use of the present invention easy to understand and understand, the technical solutions in the embodiments of the present invention will be further clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art can make various changes to the specific parameters in the present invention, but all other embodiments obtained without making creative work are within the scope of protection of the present invention.

[0047] Example 1: Expression of selenocytochrome c by selenocystine / methionine combination technology

[0048] Cytochrome c is a key structural protein. Its cysteine ​​residues (Cys14 / 17) are covalently bound to the heme prosthetic group via a thioether bond. Dynamic conformational changes (such as axially distal coordination of the heme iron) directly regulate the protein's peroxidase activity. However, existing conformational probes often act on the protein surface or are located far from the active site, making it difficult to accurately capture microenvironmental changes within the heme-binding domain. This example utilizes selenium labeling technology to develop a high-fidelity active site probe containing selenium atoms to address the shortcomings of existing technologies.

[0049] In this regard, the selenium labeling technology of cysteine ​​is used because of its similar atomic radius (Se: 1.22Å vs S: 1.04Å) and NMR sensitivity ( 77 Se chemical shift distribution reaches 2000 ppm), making it an ideal probe for analyzing the conformational dynamics in this region.

[0050] 1. Experimental Materials and Methods

[0051] 1. Source of cytochrome c: A recombinant plasmid was constructed based on the yeast cytochrome c gene sequence, expressed in E. coli, and purified using standard affinity chromatography and gel filtration methods.

[0052] 2. Selenium Labeling Strategy: The selenium-sulfur ratio in the E. coli culture medium is manipulated twice to allow for simultaneous expression and labeling of cytochrome c in E. coli. When manipulating the selenium-sulfur ratio twice, different selenocystine / methionine ratios can be used depending on the desired selenium labeling efficiency. The ratios used in both manipulations can be the same or different, and should be optimized based on the specific expression patterns of different proteins in practice. However, the general principle is that a higher selenocystine ratio results in higher selenium labeling efficiency.

[0053] 3. Specific operations for expressing selenocytochrome c using the improved method:

[0054] (1) Selection of expression system: Escherichia coli (E. coli) was selected as the expression system because of its rapid growth and easy operation.

[0055] (2) Preparation of culture medium: Prepare the culture medium by mixing 50mM Na2HPO4, 50mM KH2PO4, 50mM NH4Cl, and 10mM NaCl in a 1L conical shake flask, sterilize at high temperature, and add MgCl2, trace metal mixture, and vitamin B after cooling. 12 , vitamin mixture), glucose, calcium chloride, ampicillin and a precursor for the synthesis of heme prosthetic group (Heme).

[0056] The formula of the culture medium here is as described in Table 1 below: Table 1 Culture medium formula

[0057] .

[0058] The formula of the trace metal mixture and vitamin mixture is shown in Table 2 below: Table 2 Formulas of trace metal mixture and vitamin mixture

[0059]

[0060] (3) Expression conditions

[0061] Strain selection: Insert the cytochrome c gene into the pET series vector to obtain the expression vector, and then transform the expression vector plasmid containing the target gene (cytochrome c gene) into the host bacteria (such as Escherichia coli BL21 (DE3)) to obtain the recombinant Escherichia coli strain.

[0062] Induction conditions: Use IPTG (isopropyl-β-D-thiogalactopyranoside) as the inducer at a concentration of 1 mM. Induction temperature: 30°C. Induction time: 12-24 hours.

[0063] (4) Methods of Sulfur / Selenium Source Supplementation During Expression

[0064] During the first manipulation, 5 mM sodium sulfate is added to a 20 ml vial of culture medium as a sufficient sulfur source to ensure normal cell growth. When the culture reaches an OD value of 1.0-1.5, 1 ml of the culture medium is transferred to 1 L of fresh culture medium. During this phase, the sulfur supply is strictly limited to 50 µM sodium sulfate to help the bacteria adapt to the low-sulfur environment and establish a sulfur-starved state. This sulfur-starved state can alter the bacterial metabolic pathways, favoring the use of exogenously supplied selenocystine and methionine over internally synthesized sulfur-containing amino acids, thereby creating conditions for the synthesis of selenium-labeled proteins. During the second manipulation, when the bacteria reach the induction phase, 22.5 µM selenocystine and 5 µM methionine are added to the culture medium as sulfur / selenoamino acid precursors required for target protein synthesis. Six hours after induction, the culture medium is supplemented with the same ratio of 22.5 µM selenocystine and 5 µM methionine. This supplementation regimen follows a three-step process: a high-sulfur maintenance phase, a low-sulfur adaptation phase, and an induction supplementation phase.

[0065] Compared with Example 2, this embodiment directly adds selenocysteine ​​(selenocysteine ​​is an oxidized form of selenocysteine, which is stable in the air and can be reduced to selenocysteine ​​in solution) and methionine during the second regulation to ensure that the target protein is fully synthesized, while also meeting the selectivity of the labeling site and increasing the cysteine ​​labeling efficiency to 86% per site. This can avoid the situation where selenium is preferentially metabolized into selenomethionine and then absorbed by the protein when selenate is added. Under high selenium conditions, selenomethionine SeMet will not be converted into selenocysteine ​​SeCys in large quantities, resulting in the labeling result on methionine. Therefore, this embodiment uses the direct addition of selenocysteine ​​and methionine to avoid the low efficiency of cysteine ​​labeling, which affects the efficient and specific labeling of selenocytochrome c.

[0066] (5) Specific process of expression and purification of selenium-labeled proteins

[0067] Protein expression:

[0068] Take a portion of the prepared competent cells (stored in a -80℃ refrigerator), add the plasmid to a final concentration of 100-150ng / μL, and gently tap the bottom of the test tube to mix the contents evenly; after placing it on ice for 30 minutes, place the Eppendorf tube in a 42℃ constant temperature water bath for 90 seconds and place it on ice for 1 minute; add 500μL LB medium (without antibiotics) and culture on a shaker at 37℃ and 220rpm for 30-60 minutes; after centrifugation, re-dissolve it with 100μL of culture medium, then spread it on a plate (ampicillin resistance), and culture it upside down at 37℃ incubator overnight to allow the cells to form colonies on the plate.

[0069] On the second day, prepare the liquid culture medium (Table 1, Table 2), take 20mL of the culture medium and place it in a small conical flask. From the plate in the above step, pick a single colony and place it in a 20mL small conical flask. In the early stage of bacterial culture, add 5mM Na2SO4 as a sufficient sulfur source. Cultivate for about 13-15 hours and measure the OD 600 When the concentration is 1-1.5, transfer 1 ml of culture medium into a 1 L large flask of culture medium, incubate overnight, and supplement with 50 μM Na2SO4 to help the bacteria adapt to the low sulfur environment and establish a sulfur starvation state.

[0070] On the third day, when the bacteria grew to the induction stage, the OD 600 When the pH is about 0.75, add 22.5 μM selenocystine and 5 μM methionine as sulfur / selenium-containing amino acid precursors required for the synthesis of the target protein, maintain 37°C, 220 rpm, add inducer IPTG 1 mM after 1 hour, and culture at 30°C, 70 rpm for 6 hours, then add 22.5 μM selenocystine and 5 μM methionine again, and culture at 30°C shaking overnight.

[0071] On the fourth day, the cells were collected by centrifugation at 6000 rpm for 15 min and stored at -20°C, or directly proceeded to the next step of protein extraction and purification.

[0072] Protein purification:

[0073] The cells were collected and the culture medium was removed by centrifugation (8000 rpm, 10 min). The collected cells were resuspended in 40 mL of lysis buffer (50 mM Tris-HCl (6.057 g / L), 5 mM EDTA-2Na (1.86 g / L), pH 7.5). Lysozyme (Eggwhite, 1.5 mg / g (lysozyme / wet cells)) and 5 μl of DNA digestion enzyme (DNase I) were added and digested for 1 h. The cells were fully disrupted by high pressure and then centrifuged at 20,000 rpm for 30 min. The supernatant was placed in a clean 250 mL beaker and salted out by slowly adding ammonium sulfate (150 g / L) for 3-6 h. The supernatant was then centrifuged at 20,000 rpm for 30 min. The supernatant was dialyzed overnight (5 L: Na2HPO4 8.66 g, NaH2PO4 4.68 g, pH 7.0).

[0074] The liquid after overnight dialysis was centrifuged at 20,000 rpm for 30 min, and the supernatant was purified using an SP cation column and then molecular sieve Pg75 to collect the cytochrome c component.

[0075] In this step, the buffers A and B used for the SP cation column were: buffer A-20 mM PB (pH = 7.0), buffer B-20 mM PB and 1 M NaCl (pH = 7.0), respectively; the buffer for the molecular sieve Pg75 was 20 mM PB, 150 mM NaCl (pH = 7.0).

[0076] 4. Verification of labeling efficiency: Selenium-labeled proteins were characterized using electrospray ionization mass spectrometry (ESI-MS). The mobile phase used was acetonitrile-water (the target protein was eluted when the acetonitrile content was 50%).

[0077] 5. Control group setup: Set up unlabeled cytochrome c as the control group, and the treatment conditions are the same as those of the experimental group.

[0078] 2. Experimental results:

[0079] A bottom-up secondary mass spectrometry strategy was used to identify selenium-labeled sites in the selenocytochrome c sample. Specifically, the protein extracted from the sample undergoes reduction, alkylation, and enzymatic hydrolysis to convert it into a peptide mixture. The peptides are then electrosprayed into a mass spectrometer for scanning, generating mass spectra. Finally, these mass spectra are analyzed using relevant software to obtain relevant information about the proteins in the sample.

[0080] Detailed analysis is as follows: Mass spectrometry raw data were analyzed using MaxQuant (2.2.0.0) software, using the built-in Andromeda database search algorithm. The database used for the search was the Escherichiacoli protein sequence database downloaded from Uniprot. Search parameters were primarily default, with key parameters described below: variable modifications were set to Carbamidomethyl(C)-57.021464Da, Oxidation(M)-15.994915, Acetyl(Protein N-term)-42.010565Da, Se substitution S-47.944451Da; fixed modification was set to Carbamidomethyl(C)-57.021464Da; enzyme digestion conditions were set to Trypsin / P with a maximum of two missed cleavage sites; MS1 mass tolerances for the first and main searches were set to 20 ppm and 10 ppm, respectively; and MS2 mass tolerance was set to 20 ppm. Proteins that could not be distinguished by unique peptides were grouped together using MaxQuant software. Search results were filtered using a 1% FDR (at both protein and peptide levels).

[0081] The results showed that in the yeast cytochrome c sample obtained in this example, the proportion of single-cysteine ​​selenoprotein labeled protein was 46%, the proportion of double-cysteine ​​selenoprotein labeled protein was 40.1%, and the proportion of unlabeled protein was 13.7%. Figure 1 Verified by secondary mass spectrometry, the diselenide labeling sites were precisely located at the cysteine ​​residues (Cys14 and Cys17) ​​in cytochrome c that coordinate with the heme iron. The highest single-residue labeling efficiency reached 86%, and the target protein yield was stabilized at 1 mg / g (protein / wet cells).

[0082] like Figure 5 As shown, 50 μM selenium-labeled yeast-derived cytochrome c and 50 μM unlabeled yeast-derived cytochrome c prepared in this example were dissolved in 50 mM phosphate buffer and 100 μM potassium ferrocyanide was added to prepare them into an oxidized state (OX). At the same time, 50 μM selenium-labeled yeast-derived cytochrome c and 50 μM unlabeled yeast-derived cytochrome c prepared in this example were dissolved in 50 mM phosphate buffer and 100 μM ascorbic acid was added to prepare them into a reduced state (re). The spectra were detected on a circular dichroism spectrometer. The results showed that the selenium-labeled cytochrome c prepared in this example was almost indistinguishable from the original wild-type cytochrome c in either the oxidized or reduced state, indicating that the method of this example can be used for the study of biomolecular structures.

[0083] like Figure 7 As shown in the figure, the cytochrome c of selenocysteine ​​prepared in this example at a concentration of 1.5 mM is 77 The SeNMR spectrum, with a sampling time of 9h30min, clearly shows the signal of selenium atoms.

[0084] like Figure 8 As shown in the figure, the two-dimensional structure of cytochrome c of selenocysteine ​​prepared in this example in the oxidized and reduced states is shown in the figure. 1 H- 77 Se HSQC correlation spectrum shows that the selenium atoms introduced into the protein by the present invention can serve as a probe to react to changes in protein conformation.

[0085] The above experimental results show that the selenium labeling technology of the present invention has successfully achieved specific selenium labeling of cysteine ​​residues in cytochrome c, with a single-site labeling efficiency of 86% and a protein yield of 1 mg / g (protein / wet weight of bacteria). The conformation of the labeled protein remains essentially unchanged, providing a high-fidelity probe for the conformational study of protein active sites centered on heme. However, the traditional selenium-sulfur inorganic salt replacement method of Example 2 has significant nonspecific labeling (methionine sites account for >70%, Figure 3 ), the 20 amino acid replacement rules in Example 3 had a labeling efficiency of less than 40% due to interference from endogenous cysteine ​​synthesis (LC-MS / MS verification, Figure 4 ).

[0086] Example 2: Selenocytochrome c expressed by selenium-sulfur inorganic salt substitution method

[0087] 1. Experimental Materials and Methods

[0088] 1. According to the reported method of adding inorganic selenium and sulfur salts, the specific steps for expressing and purifying selenium cytochrome c are as follows:

[0089] Protein expression:

[0090] Take an aliquot of the prepared BL21 competent cells (stored in a -80°C refrigerator), add the same yeast-derived cytochrome c recombinant plasmid as in Example 1, with a final plasmid concentration of 100-150 ng / μL, and gently tap the bottom of the test tube to mix the contents evenly; after placing on ice for 30 minutes, place the Eppendorf tube in a 42°C constant temperature water bath for 90 seconds and place on ice for 1 minute; add 500 μL of LB medium (without antibiotics) and incubate at 37°C at 220 rpm for 30-60 minutes; after centrifugation, re-dissolve with 100 μL of culture medium, then spread on a plate (ampicillin resistance), and incubate in an incubator at 37°C overnight to allow cells to form colonies on the plate.

[0091] On the second day, prepare the liquid culture medium (Table 1) and take 20 mL of the culture medium into a small conical flask. Pick a single colony from the plate in the above step and place it into the 20 mL small conical flask. In the early stage of bacterial culture, add 5 mM Na2SO4 as a sufficient sulfur source. Cultivate for about 13-15 hours and measure the OD 600 When the concentration of sulfur is 1-1.5, 1 ml of culture medium is transferred to a 1 L large bottle of culture medium, culture overnight and supplemented with 50 μM Na2SO4 to enable the bacteria to adapt to the low sulfur environment and establish a sulfur starvation state.

[0092] On the third day, when the bacteria grew to the induction stage, the OD 600 When the C-value is about 0.75, add sodium selenite and sodium sulfate in a specific ratio, maintain at 37℃, 220rpm, add 1mM inducer after 1h, and culture at 30℃, 70rpm for 6h, then add sodium selenite and sodium sulfate in the same ratio again, and culture at 30℃ shaking overnight.

[0093] On the fourth day, the cells were collected by centrifugation at 6000 rpm for 15 min and stored at -20°C, or directly proceeded to the next step of protein extraction and purification.

[0094] Protein purification:

[0095] The cells were collected and the culture medium was removed by centrifugation (8000 rpm, 10 min). The collected cells were resuspended in 40 mL of lysis buffer (50 mM Tris-HCl (6.057 g / L), 5 mM EDTA-2Na (1.86 g / L), pH 7.5). Lysozyme (Eggwhite, 1.5 mg / g (lysozyme / wet cells)) and 5 μl of DNA digestion enzyme (DNase I) were added and digested for 1 h. The cells were fully disrupted by high pressure and then centrifuged at 20,000 rpm for 30 min. The supernatant was placed in a clean 250 mL beaker and salted out by slowly adding ammonium sulfate (150 g / L) for 3-6 h. The supernatant was then centrifuged at 20,000 rpm for 30 min. The supernatant was dialyzed overnight (5 L: Na2HPO4 8.66 g, NaH2PO4 4.68 g, pH 7.0).

[0096] The liquid after overnight dialysis was centrifuged at 20,000 rpm for 30 min, and the supernatant was purified using an SP cation column and then molecular sieve Pg75 to collect the cytochrome c component.

[0097] In this step, the buffers A and B used for the SP cation column were: buffer A-20 mM PB (pH = 7.0), buffer B-20 mM PB and 1 M NaCl (pH = 7.0), respectively; the buffer for the molecular sieve Pg75 was 20 mM PB, 150 mM NaCl (pH = 7.0).

[0098] 2. Verification of labeling efficiency: Selenium-labeled proteins were characterized using electrospray ionization mass spectrometry (ESI-MS). The mobile phase used was acetonitrile-water (the target protein was eluted when the acetonitrile content was 50%).

[0099] 3. Control group setting: Set up unlabeled cytochrome c as the control group, and the treatment conditions are the same as those of the experimental group.

[0100] 2. Experimental Results

[0101] In this experiment, two groups of ratios were made to observe the labeling efficiency, such as Figure 2 , where the ratio of selenium to sulfur inorganic salts is 0.5 (i.e. 1:1), it means that the sodium selenite and sodium sulfate added to the culture medium are both 25 μM, and where the ratio of selenium to sulfur inorganic salts is 0.9, it means that the sodium selenite and sodium sulfate added to the culture medium are 22.5 μM and 5 μM, respectively. After mass spectrometry analysis, it was found that the primary mass spectrometry showed a higher labeling efficiency when the ratio of selenium to sulfur inorganic salts was 0.9 (i.e. 9:1).

[0102] Therefore, yeast-derived cytochrome c expressed with a 0.9 ratio of selenium to sulfur inorganic salts was subjected to secondary mass spectrometry to identify selenium-labeled sites. The method was the same as described in Example 1. The final analysis revealed that selenium-labeled sites accounted for 72.83% of the protein at methionine 80 and 9.68% at methionine 64 (Figure 3). Cysteines 14 and 17, which are stably linked to the heme in the active site, were not labeled with selenium.

[0103] Example 3: Selenocytochrome c expressed by adding 20 amino acids

[0104] 1. Experimental Materials and Methods

[0105] 1. Sulfur / selenium source supplementation method: According to literature reports, the main difference between this method and the selenium-sulfur inorganic salt addition method is that there is no strict restriction on the sulfur source in the culture medium. Selenium labeling mainly involves large-scale supplementation of selenocystine and other amino acids required for protein synthesis during the protein expression stage, so that the protein is selenium-labeled during the synthesis process.

[0106] 2. The specific experimental steps are as follows:

[0107] Protein expression:

[0108] Take an aliquot of the prepared BL21 competent cells (stored in a -80°C refrigerator), add the same yeast-derived cytochrome c recombinant plasmid as in Example 1, with a final plasmid concentration of 100-150 ng / μL, and gently tap the bottom of the test tube to mix the contents evenly; after placing on ice for 30 minutes, place the Eppendorf tube in a 42°C constant temperature water bath for 90 seconds and place on ice for 1 minute; add 500 μL of LB medium (without antibiotics) and incubate at 37°C at 220 rpm for 30-60 minutes; after centrifugation, re-dissolve with 100 μL of culture medium, then spread on a plate (ampicillin resistance), and incubate in an incubator at 37°C overnight to allow cells to form colonies on the plate.

[0109] On the next day, a single clone was picked and cultured in 1 ml LB at 37°C and 220 rpm in a shaking incubator for about 12 h.

[0110] 3. Prepare the culture medium (stage 1) by mixing 25mM Na2HPO4, 25mM KH2PO4, 50mM NH4Cl, and 5mM Na2SO4 in a 1L conical shake flask, sterilize at high temperature, and add MgSO4, trace metal mixture, and vitamin B after cooling. 12 , vitamin mixture (as shown in Table 2), 10mM glucose, ampicillin and the precursor of heme prosthetic group synthesis (Heme). Take 100ml of this culture medium and culture it in a small conical flask for 4-6 hours, then transfer it to 1L culture medium and culture it until the OD 600=1-1.5, add the ingredients in stage 2, which consist of 10mM glucose, aspartate, 17 amino acids, methionine and selenocysteine. o C, incubate at 220 rpm for 10 min, then change to 30 o C, 70 rpm, after 10 min of recovery, 1 mM IPTG was added to initiate induction and expression was allowed to proceed overnight.

[0111] The formula of the culture medium here is as described in Table 3 below: Table 3 Culture medium formula

[0112]

[0113] The formula of the trace metal mixture in the above table is shown in Table 4 below: Table 4 Formula of trace metal mixture

[0114]

[0115] On the third day, centrifuge at 6000 rpm for 15 min, discard the supernatant, collect the bacteria and store at -20°C, or proceed directly to the next step of protein extraction and purification.

[0116] The subsequent protein purification steps were the same as those in Examples 1 and 2.

[0117] 2. Experimental Results

[0118] This experiment tried two groups of induced expression conditions, respectively at OD 600 When the concentration is 1 and 1.5, add amino acids. Figure 4 As shown in Figure 2, the protein selenium labeling efficiency obtained in both experiments was very low as verified by mass spectrometry. 600 The expression group in which amino acids were added when the expression ratio was 1.5 had a slightly higher final labeling efficiency, but still less than 40%.

[0119] Example 4 Selenium Labeling of Active Proteins Containing Free Sulfhydryl Groups (-SH)

[0120] Ubiquitin (Ub) is a highly conserved eukaryotic regulatory protein (76 amino acids, molecular weight 8.451 kDa). Through ubiquitination, it regulates diverse biological processes, including protein degradation, cell cycle regulation, and signal transduction. Its core function is achieved through the ubiquitination pathway. Polyubiquitin chains at positions K48, K63, and K11 are recognized by the 26S proteasome. Ubiquitin receptors (such as UBR1) on the regulatory subunit (19S) specifically bind to these chains, driving substrate hydrolysis by the 20S core subunit (ΔG = -7.3 kcal / mol) under ATPase energy. Monoubiquitination can mediate membrane protein endocytosis and vesicular trafficking, such as EGFR internalization. Studies have shown that a ubiquitin mutant with a K63-to-cysteine ​​mutation (K63C) blocks polyubiquitin chain formation (reducing K48 / K63 heterotypic ligation efficiency by >95%), providing an ideal model for studying the dynamics of monoubiquitination.

[0121] In this example, the selenium labeling technology of the present invention is used to specifically label the ubiquitin K63C mutant at the mutation site, so that 77 The high sensitivity of Se NMR is used to study the structure-activity relationship of ubiquitin.

[0122] 1. Experimental Materials and Methods

[0123] 1. Source of the ubiquitin K63C mutant: The K63 position of wild-type ubiquitin was mutated to cysteine ​​via site-directed mutagenesis. The ubiquitin K63C mutant was expressed in Escherichia coli using IPTG induction, cultured at 37°C for 4 hours, and purified by affinity chromatography and gel filtration.

[0124] 2. Expression and purification of selenium-tagged ubiquitin K63C mutant:

[0125] Protein expression:

[0126] Take a portion of the prepared BL21 competent cells (stored in a -80°C refrigerator), add the constructed ubiquitin K63C mutant plasmid to a final concentration of 100-150 ng / μL, and gently tap the bottom of the test tube to mix the contents evenly; after placing it on ice for 30 minutes, place the Eppendorf tube in a 42°C constant temperature water bath for 90 seconds and place it on ice for 1 minute; add 500 μL LB medium (without antibiotics) and culture on a shaker at 37°C and 220 rpm for 30-60 minutes; after centrifugation, re-dissolve it with 100 μL of culture medium, then spread it on a plate (ampicillin resistance), and culture it upside down at 37°C incubator overnight to allow the cells to form colonies on the plate.

[0127] On the second day, prepare the liquid culture medium (Table 1, Table 2), take 20mL of the culture medium and place it in a small conical flask. Pick a single colony from the plate in the above step and place it in the 20mL small conical flask. In the early stage of bacterial culture, add 5mM Na2SO4 as a sufficient sulfur source. Cultivate for about 13-15 hours and measure the OD 600 When the concentration of sulfur is 1-1.5, 1 ml of culture medium is transferred to a 1 L large bottle of culture medium, culture overnight and supplemented with 50 μM Na2SO4 to enable the bacteria to adapt to the low sulfur environment and establish a sulfur starvation state.

[0128] On the third day, when the bacteria grew to the induction stage, the OD 600 When the pH is around 0.75, add 22.5 μM selenocystine and 5 μM methionine as sulfur / selenium amino acid precursors required for target protein synthesis. Maintain at 37°C, 220 rpm for 1 hour. After that, add 1 mM IPTG inducer and incubate at 37°C for 4 hours. Then, add 22.5 μM selenocystine and 5 μM methionine again. Incubate at 37°C on a shaker for 4 hours before harvesting the cells. Harvest the cells by centrifugation at 6000 rpm for 15 minutes and store at -20°C or proceed directly to the next step of protein extraction and purification.

[0129] Protein purification:

[0130] The collected bacteria were resuspended in 20mM sodium acetate (pH=5.0), 3mM DTT, and high pressure was applied to fully break the bacteria, and then centrifuged at 20000rpm for 30min; the supernatant after centrifugation was placed in a clean 250ml beaker and separated and purified by SP cation column. The buffer used was buffer A-20mM sodium acetate, 3mM DTT, pH=5.0; buffer B-20mM sodium acetate, 1M NaCl, 3mM DTT, pH=5.0. Using gradient separation, the target protein was eluted at 30% buffer B. The target protein fraction was collected and concentrated to 4mL, and then further purified using molecular sieve Pg75. The buffer used was the above buffer A, and the target protein fraction was eluted at 60mL. The target protein was collected and placed in 4 o C short-term storage.

[0131] 3. Control group setting: Set up the unlabeled ubiquitin K63C mutant as the control group, and the treatment conditions are consistent with those of the experimental group.

[0132] 2. Experimental results:

[0133] After primary mass spectrometry identification, the selenium labeling method of the present invention successfully achieved specific selenium labeling of the ubiquitin K63C mutant at cysteine ​​63, with a labeling efficiency of 64%. Figure 6As shown, 8540Da is the unlabeled molecular weight of the protein, and 8600Da is the molecular weight of the protein after selenium labeling. After selenium labeling, a methyl group is bound to the corresponding site, so there is an increase of 60Da (pure selenium labeling is an increase of 46Da).

Claims

1. A method for achieving efficient selenium labeling of proteins, characterized in that: A plasmid expressing the target protein is transferred into a prokaryotic expression system. During bacterial culture, the ratio of selenium and sulfur in the culture medium, which are nutrients related to protein synthesis, is manipulated twice, allowing the target protein to be expressed and labeled simultaneously in the prokaryotic expression system. The first manipulation involves establishing an adequate sulfur source environment during the initial bacterial growth phase, followed by a gradual reduction in the sulfur source concentration to induce a sulfur starvation state. The second regulation includes supplementing the culture medium with natural sulfur-containing amino acids and amino acids that replace sulfur with selenium, which are related to the synthesis of target proteins, twice when the bacteria grow to the induction stage. The ratio of the amino acids that replace sulfur with selenium to the natural sulfur-containing amino acids is 1 to 9:

1.

2. The method for achieving efficient selenium labeling of proteins according to claim 1, wherein The target protein is a biological macromolecule formed by connecting amino acids with peptide bonds, including small peptides, polypeptides, peptide segments, proteins, and complexes of proteins and peptides.

3. The method for achieving efficient selenium labeling of proteins according to claim 1, wherein The target protein is cytochrome c.

4. The method for achieving efficient selenium labeling of proteins according to claim 1, wherein The prokaryotic expression system is an Escherichia coli expression system, the natural sulfur-containing amino acid is methionine, and the amino acid in which selenium replaces sulfur is selenocystine.

5. The method for achieving efficient selenium labeling of proteins according to claim 4, wherein In the second regulation, the molar concentration ratio of selenocysteine ​​to methionine added to the culture medium for the first and second times is 4.5:

1.

6. The method for achieving efficient selenium labeling of proteins according to claim 5, wherein: The preparation of the cytochrome c containing selenium-labeled cysteine ​​specifically comprises the following steps: (1) Expression of Selenium-labeled target protein: First, the target protein plasmid was transferred into E. coli, and the transferred E. coli was evenly spread on LB solid medium and cultured in a 37°C incubator overnight; On the second day, prepare the liquid culture medium and take 20 ml of the culture medium into a small conical flask. Pick a single clone from the plate in the above step and add 5mM Na2SO4 into the 20 ml small conical flask. Incubate for about 13-15 hours and measure the OD 600 When the concentration is 1-1.5, transfer 1 ml into a 1 L flask of culture medium overnight and supplement with 50 μM Na2SO4; On the third day, measure OD 600 When the pH value is about 0.75, add 45 μM selenocystine and 5 μM methionine for the first time, maintain at 37°C, 220 rpm, add 1 mM inducer after 1 hour, and culture at 30°C, 70 rpm for 6 hours, and then add selenocystine and methionine in the same proportion as above; Culture overnight until the fourth day, harvest the cells by centrifugation at 6000 rpm for 15 min, and store at -20°C or proceed directly to the next step of protein extraction and purification; (2) Purification of cytochrome c labeled with selenium and cysteine: Take out the bacteria that have just been centrifuged or stored at -20℃, resuspend them in 40mL lysis buffer (50mM Tris-HCl (6.057g / L), 5mM EDTA-2Na (1.86g / L), pH7.5), add lysozyme ( Eggwhite, 1.5 mg / g (lysozyme / wet bacteria)) and DNA digestion enzyme (DNaseⅠ) 1 μl / ml (bacterial liquid volume) were digested for 1 hour, and then high pressure was applied to fully disrupt the bacteria, followed by centrifugation at 20,000 rpm for 30 minutes; the supernatant after centrifugation was placed in a clean 250 ml beaker, and ammonium sulfate (150 g / L) was slowly added for salting out for 3-6 hours, followed by centrifugation at 20,000 rpm for 30 minutes, and the supernatant was dialyzed overnight (5 L: Na2HPO4 8.66 g, NaH2PO4 4.68 g, pH 7.0); the liquid after overnight dialysis was centrifuged at 20,000 rpm for 30 minutes, and the supernatant was purified using an SP cation column and then a molecular sieve Pg75 to collect the cytochrome c component.

7. The method for achieving efficient selenium labeling of proteins according to claim 6, wherein: The preparation method of the liquid culture medium in step (1) is as follows: 50mM Na2HPO4, 50mM KH2PO4, 50mM NH4Cl, and 10mM NaCl are placed in a 1L conical shake flask, sterilized at high temperature, and after cooling, 2mM MgCl2, a trace metal mixture, a vitamin mixture, 0.4% glucose, 200μM calcium chloride, 100μg / mL ampicillin antibiotic, and a precursor for synthesizing heme prosthetic groups are added.

8. The method for achieving efficient selenium labeling of proteins according to claim 6, wherein: In step (2), the buffers A and B used for the SP cation column are: buffer A is 20 mM PB (pH = 7.0), and buffer B is 20 mM PB and 1 M NaCl (pH = 7.0); the buffer for the molecular sieve Pg75 is 20 mM PB, 150 mM NaCl (pH = 7.0).

9. A cytochrome c labeled with cysteine ​​prepared by the method according to any one of claims 3 to 8, characterized in that: The specific selenium labeling efficiency of the cysteine ​​residues in the cytochrome c reaches 86%, and the protein yield is increased to 1 mg / g.

10. Cytochrome c of the selenium-labeled cysteine ​​according to claim 9 in selenium nuclear magnetic resonance (S-NMR) 77 Se NMR) in protein conformational characterization.

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