A method for efficient selenium labeling of proteins

Abstract

The application discloses a method for efficiently labeling proteins with selenium, which comprises the following steps: adjusting the ratio of selenium and sulfur in the culture medium during the expression of a plasmid containing a target protein by a prokaryotic expression system, and realizing the efficient replacement of selenium atoms for the cysteine sulfur atoms in the target protein; under the premise of maintaining the good growth state of cells, the selenium labeling efficiency can reach 86%, and the protein expression amount is stably about 1 mg / g (protein / biomass wet weight); structure analysis shows that the obtained selenium labeled protein is basically consistent with the natural protein in the secondary structure and does not change obviously. The method is simple in operation, high in labeling efficiency and protein yield, good in repeatability, and suitable for structural biology research such as nuclear magnetic resonance, provides an effective means for studying the function mechanism of sulfur atoms in biological macromolecules (including small peptides, polypeptides, peptide segments, proteins, and complexes of proteins or peptides) by using selenium as a probe, and has good scientific research application prospect and industrialization popularization value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for achieving efficient selenium labeling of proteins, namely, a method for efficiently replacing sulfur atoms of cysteine ​​in proteins with selenium atoms. Background Technology

[0002] Sulfur plays a vital role in living organisms, especially cysteine ​​(Cys). As a key component of the active site of proteins, Cys is widely present in various organisms and its concentration increases with the evolutionary level of species, indicating its important role in the regulation of higher biological structures. The main functions of cysteine ​​include promoting proper protein folding, regulating redox states, and participating in the functional regulation of active sites. However, existing methods for determining protein structure, such as mass spectrometry, spectroscopy, X-ray diffraction, and nuclear magnetic resonance (NMR), cannot directly detect sulfur atoms, thus limiting the study of the mechanisms by which sulfur atoms participate in biological processes.

[0003] Selenium atoms, due to their similar chemical properties to sulfur atoms, have become a target for utilizing selenium nuclear magnetic resonance (NMR). 77 Se NMR is an ideal alternative for detecting sulfur atoms. 77 Se isotopes, with a nuclear spin quantum number of 1 / 2 and a natural abundance of 7.6%, are suitable for NMR detection. Studies have shown that selenocysteine ​​(Sec) not only possesses unique chemical properties, such as a lower pKa value that facilitates deprotonation for nucleophilic reactions, but its larger atomic radius also endows it with unique reaction characteristics under specific conditions. Early X-ray crystallography studies found that selenium labeling has minimal impact on protein structure, confirming its feasibility as an interference-free labeler. Furthermore, 77 Se NMR technology has made significant progress in recent years, with a chemical shift range of up to 2000 ppm. It exhibits high sensitivity to the environment and is suitable for accurate characterization of complex systems.

[0004] For the study of sulfur-containing proteins, such as cytochrome c (CYT c), the active site is covalently linked to the peptide chain via a heme prosthetic group through a cysteine ​​side chain, exhibiting different coordination states under oxidative stress. Traditional characterization methods such as mass spectrometry and Raman spectroscopy can reveal the coordination state, but they cannot directly reflect the dynamic conformational changes. In contrast, NMR technology, due to its ability to dynamically monitor structural changes, demonstrates unique advantages in resolving the relationship between protein function and structure. Introducing selenocysteine ​​into the active site of CYT c allows for the utilization of... 77 Se NMR technology can monitor the conformational changes of cyt c active sites in biological processes in real time, thereby gaining a deeper understanding of the relationship between its structure and function.

[0005] However, to achieve utilization77 The first step in studying the structure-activity relationship of proteins related to sulfur atoms by Se NMR technology is to prepare selenium-labeled protein samples. The existing methods for selenium-labeled proteins, such as selenium-sulfur inorganic salt substitution, 20 kinds of amino acid addition, C321 system amber codon expression, etc., have low labeling efficiency, unstable yield, and cannot specifically label the sulfur atoms of cysteine, so they cannot be applied to 77 Se NMR detection. Therefore, it is particularly urgent to develop an efficient method for preparing selenium-labeled proteins and to simultaneously distinguish between cysteine and methionine sites. SUMMARY

[0006] Based on the Escherichia coli expression system, the present application successfully realizes specific selenium labeling of the sulfur atoms of cysteine residues in proteins by precisely regulating the supply ratio of sulfur element and selenium element content in the culture medium, providing key technical support for directly analyzing the molecular mechanism of cysteine in protein conformation and functional regulation using nuclear magnetic resonance (NMR) technology. The core of the technology is based on the similarity of the physicochemical properties of selenium atoms and sulfur atoms (atomic radius difference < 0.2 Å), and by establishing a dynamic balance 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, nuclear magnetic level selenium-labeled protein samples are obtained through high-density fermentation, combined with 77 Se NMR spectrum analysis, the structural dynamic changes of the cysteine site can be accurately tracked.

[0007] This scheme significantly reduces the toxic effects of selenium elements on host cells (cell survival rate is increased to 82%) by optimizing multiple parameters such as amino acid metabolic balance, operational feasibility, target protein yield (up to 1 mg / L), and labeling efficiency (86%). The expression system and plasmid design are universal and can be widely applied to various cysteine-containing protein systems (including free thiol and proteins involved in sulfur coordination bonds), significantly expanding the compatibility boundary of selenium labeling technology.

[0008] The method for preparing selenium-labeled cytochrome c of the present application comprises the following steps:

[0009] First, expression of the selenium-labeled target protein:

[0010] 1. Transformation:

[0011] The plasmid of the target protein is transformed into Escherichia coli, and the transformed Escherichia coli is uniformly coated on LB solid medium and cultured in a 37°C incubator overnight.

[0012] Take a prepared competent (stored in -80 °C refrigerator), add plasmid final concentration of 100-150 ng / μL, gently hit the bottom of the test tube to mix the contents, place the Ep tube in a 42 °C constant temperature water bath for 90 s, place on ice for 1 min, add 500 μL LB (without antibiotics), 37 °C, 220 rpm shaker for 30-60 min, centrifuge and resuspend with 100 μL medium to plate (ampicillin resistance), invert in a 37 °C incubator overnight.

[0013] 2. Pick single:

[0014] Prepare the medium, 50 mM Na2HPO4, 50 mM KH2PO4, 50 mM NH4Cl, 10 mM NaCl, in a 1 L conical flask, autoclave, cool down, add MgCl2, trace metal mixture, vitamin mixture, vitamin B 12、 glucose, calcium chloride, ampicillin, and precursors of heme synthesis.

[0015] 3. Expression:

[0016] Take 20 mL of the medium into a small conical flask, pick a single colony from the plate in the above step into the 20 mL small conical flask and supplement with 5 mM Na2SO4, culture for about 13-15 h, measure OD to be 1-1.5, take 1 mL into a 1 L flask medium overnight and supplement with 50 μM Na2SO4, the next day when the OD is about 0.75, add the first 22.5 μM selenocysteine and 5 μM methionine, maintain at 37 °C, 220 rpm, 1 h later add the inducer 1 mM, and culture at 30 °C, 70 rpm for 6 h, then supplement with the same proportion of selenocysteine and methionine. Centrifuge at 6000 rpm for 15 min to collect the bacteria overnight.

[0017] Second step, purification method of selenolabelled cytochrome c:

[0018] 1. Take out the bacteria that have been centrifuged to remove the supernatant or stored at -20 °C, resuspend in 40 mL lysis buffer (50 mM Tris-HCl (6.057 g / L), 5 mM EDTA-2Na (1.86 g / L), pH 7.5), add lysozyme (Eggwhite, 1.5 mg / g lysozyme / wet bacterial body) and 5 μL DNA digestion enzyme (DNase I) for 1 h.

[0019] 2. Use high pressure to repeatedly break the bacterial body, then centrifuge at 20000 rpm for 30 min.

[0020] 3. The supernatant after centrifugation was placed in a clean 250ml beaker, and ammonium sulfate (150g / L) was slowly added for salting out for 3-12h.

[0021] 4. Centrifugation at 20000rpm for 30min, take the supernatant and dialysis overnight (5L: Na2HPO4 8.66g, NaH2PO4 4.68g, pH7.0).

[0022] 5. The liquid after overnight dialysis was centrifuged at 20000rpm for 30min, and the precipitate was removed before column purification.

[0023] 6. SP cation column purification.

[0024] 7. Molecular sieve pg75 column purification.

[0025] Third step, protein spectrum identification:

[0026] The selenium-labeled protein is characterized by using electrospray ionization mass spectrometry (ESI-MS) technology. The mobile phase is selected as an acetonitrile-water system (the target protein is eluted when the proportion of acetonitrile is 50%). Taking selenium-labeled cytochrome c as an example, the results show that the proportion of single-cysteine selenium-labeled protein is 46%, the proportion of double-cysteine selenium-labeled protein is 40.1%, and the proportion of non-selenium-labeled protein is 13.7%. Through secondary mass spectrometry verification, the double-selenium labeling site is accurately located in the cysteine residues (Cys14 / 17) in cytochrome c which are coordinated with the heme iron, and the highest single-residue labeling efficiency reaches 86%, and the yield of the target protein is stably at 1mg / g (protein / wet microbial body).

[0027] The application also provides the application of the selenium-labeled cytochrome c with selenium-labeled cysteine prepared by the above method in protein conformation characterization by Se NMR. 77 The application further provides the application of the selenium-labeled cytochrome c with selenium-labeled cysteine prepared by the above method in protein conformation characterization by Se NMR.

[0028] Compared with the prior art, the application has the following beneficial effects:

[0029] 1. The current selenium-labeled protein preparation technology mainly has the following limitations:

[0030] (1) Chemical modification method: coupling selenium-containing small molecules to proteins through chemical reaction, which is simple to operate but only suitable for sites containing free sulfhydryl groups. Exogenous modification groups easily cause steric hindrance effect (alpha-helix twist degree >= 15%), which interferes with the natural conformation of the protein;

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

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

[0033] 1) Selenium-sulfur inorganic salt substitution method ( Figure 2 , Figure 3 ): By gradually replacing the sulfate (SO4) in the culture medium 2- ) is selenite (SeO3) 2- Selenium labeling was achieved, but mass spectrometry analysis showed that the selenium labeling sites were mainly sulfur atoms in methionine, and specific selenium labeling could not be performed on cysteine.

[0034] 2) Method of adding 20 amino acids separately ( Figure 4 ): Twenty amino acids were added to the culture medium, with selenocysteine ​​directly replacing natural cysteine. Although cysteine ​​site-specific labeling could be achieved, the labeling efficiency was low.

[0035] 3) Modify the methionine labeling method: (Referencing...) 13 C-methionine isotope labeling strategy, but mass spectrometry results showed that labeling was unsuccessful;

[0036] 4) C321 Amber Codon System: Based on non-natural amino acid insertion technology using stop codon reprogramming, this system requires the co-expression of six accessory proteins, including SelD and CCHL, and depends on the C321ΔRF1 genetically engineered strain. This system is complex to construct, has demanding expression conditions, and results in low yields of the target protein.

[0037] 2. The method proposed in this invention overcomes the above problems. Compared with the methods mentioned above, this invention is simple to operate, has a selenium labeling efficiency of up to 86%, and produces high and stable protein yields of 1 mg / g (protein / wet bacterial cell). It can also achieve specific labeling of cysteine ​​sites. Furthermore, the method of this invention has good compatibility and universality, and can be used to fill the gaps in the study of the functional mechanisms of sulfur atoms in biological systems, thus possessing high scientific research application value. Attached Figure Description

[0038] Figure 1 The mass spectrometry identification results of cysteine ​​selenium-labeled cytochrome c prepared in Example 1 show that cytochrome c labeled with selenium at single cysteine ​​sites (se-ycc) accounted for 46%, and cytochrome c labeled with selenium at double cysteine ​​sites (se2-ycc) accounted for 40.1%, thus the percentage of selenium-labeled single cysteine ​​sites reached 86.1%. ycc represents unlabeled cytochrome c.

[0039] Figure 2The results are the first-order mass spectrometry statistics of selenocytochrome c expressed by the selenium-sulfur inorganic salt substitution method in Example 2. The left side shows the results of selenocytochrome c expressed when the ratio of selenium-sulfur inorganic salt is 0.5 (1:1), and the right side shows the results of selenocytochrome c expressed when the ratio of selenium-sulfur inorganic salt is 0.9 (9:1).

[0040] Figure 3 For the secondary mass spectrometry analysis of selenium cytochrome c expressed by the selenium-sulfur inorganic salt substitution method in Example 2, it was found that the selenium labeling site accounted for 72.83% at position 80 of methionine and 9.68% at position 64 of methionine.

[0041] Figure 4 The mass spectrometry structures of selenium cytochrome c expressed by the addition of 20 amino acids in Example 3 are shown. They were analyzed at different OD values. 600 Add amino acids (OD on the left) 600 =1, the right side is OD 600 =1.5), the efficiency of selenium labeling even in OD 600 Even at 1.5, it is still less than 50%.

[0042] Figure 5 To confirm that the selenium-labeled protein expressed by the novel method in Example 1 has no effect on the protein structure, the oxidized (OX) and reduced (re) state samples of selenium-labeled cytochrome c prepared in Example 1 were compared with wild-type cytochrome c samples. The circular dichroism chromatogram results showed that the prepared selenium-labeled cytochrome c had almost no structural difference from the original wild-type cytochrome c, indicating that the method in Example 1 can be used for biomolecular structure research.

[0043] Figure 6 The mass spectrum of the ubiquitin protein (se-UB) of selenocysteine ​​expressed in Example 4 is shown. 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, the molecular weight increases by 60 Da because a methyl group is bound to the corresponding site (the increase is 46 Da for selenium labeling alone).

[0044] Figure 7 One-dimensional cytochrome c of selenium-labeled cysteine ​​prepared in Example 1 at a concentration of 1.5 mM. 77 Se NMR spectrum, sampled for 9 hours and 30 minutes, clearly shows the signal of selenium atoms.

[0045] Figure 8 Two-dimensional images of cytochrome c of selenocysteine ​​prepared in Example 1 in oxidized and reduced states. 1 H, 77 Se-HSQC correlation spectroscopy indicates that the selenium atoms introduced into the protein in this invention can act as probes to detect changes in protein conformation. Detailed Implementation

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

[0047] Embodiment 1: Expression of selenium cytochrome c by selenium cysteine / methionine combination technology

[0048] Cytochrome c is a key structural protein, and its cysteine residues (Cys14 / 17) are covalently bound to the heme prosthetic group through a thioether bond. Its dynamic conformational change (such as heme iron axial distal coordination) directly regulates the peroxidase activity of the protein. However, most existing conformational probes act on the surface of the protein or are far from the active center, and it is difficult for existing conformational probes to accurately capture the microenvironment changes of the heme binding domain. This embodiment develops a high-fidelity probe containing selenium atoms in the active center by selenium labeling technology to solve the shortcomings of the prior art.

[0049] For this reason, selenium labeling technology of cysteine, due to its atomic radius similarity (Se: 1.22 Å vs S: 1.04 Å) and nuclear magnetic resonance sensitivity (Se chemical shift distribution up to 2000 ppm), becomes an ideal probe for analyzing the conformational dynamics of this region. 77 Se chemical shift distribution up to 2000 ppm), becomes an ideal probe for analyzing the conformational dynamics of this region.

[0050] I. Experimental materials and methods:

[0051] 1. Source of cytochrome c: Recombinant plasmid is constructed according to the gene sequence of yeast-derived cytochrome c, expressed by E. coli system, and obtained after standard affinity chromatography and gel filtration purification.

[0052] 2. Selenium labeling strategy: The selenium-sulfur element ratio in the E. coli culture medium is regulated twice, so that cytochrome c is expressed and labeled at the same time during expression in E. coli. When regulating the selenium-sulfur element ratio twice, different selenium cysteine / methionine ratios can be used according to the desired selenium labeling efficiency, and the ratios used in the two regulations can be the same or different. It should be optimized according to the actual operation of different proteins in different expression conditions. However, the general principle is that the greater the proportion of selenium cysteine, the higher the selenium labeling efficiency.

[0053] 3. The detailed operation of expressing selenium cytochrome c using the improved method:

[0054] (1) Selection of expression system, E. coli is selected as the expression system because of its fast growth and easy operation.

[0055] (2) Preparation of culture medium, configure the culture medium, 50mM Na2HPO4, 50mM KH2PO4, 50mM NH4Cl, 10mM NaCl are configured in 1L conical flask, high temperature sterilization, after cooling, MgCl2, trace metal mixture, vitamin B 12 , vitamin mixture), glucose, calcium chloride, ampicillin and synthetic heme precursor.

[0056] The formula of the culture medium is shown in Table 1 below:

[0057] Table 1 Formula of culture medium

[0058] .

[0059] The formula of trace metal mixture and vitamin mixture is shown in Table 2:

[0060] Table 2 Formula of trace metal mixture and vitamin mixture

[0061]

[0062] (3) Expression conditions

[0063] Strain selection: insert cytochrome c gene into pET series vector, obtain expression vector, transfer the expression vector plasmid containing the target gene (cytochrome c gene) into host bacteria (such as E. coli BL21 (DE3)), obtain recombinant E. coli strain.

[0064] Induction conditions: IPTG (isopropyl-β-D-thiogalactoside) is used as the inducer, the concentration is 1mM. Induction temperature: 30℃. Induction time: 12-24 hours.

[0065] (4) Sulfur / selenium source supplementing method during expression

[0066] The first regulation is to add 5 mM sodium sulfate as a sufficient sulfur source in 20 ml of small vial medium to ensure normal growth of the cells at the beginning of bacterial culture. When the culture grows to OD = 1.0-1.5, 1 ml of the culture is transferred to 1 L of fresh medium. At this stage, the supply of sulfur source is strictly limited, and only 50 µM sodium sulfate is supplemented to adapt the bacteria to a low-sulfur environment and establish a sulfur-starved state, which can induce changes in the metabolic pathways of the bacteria, making them more inclined to use exogenously provided selenocysteine and methionine instead of self-synthesized sulfur-containing amino acids, thereby creating conditions for the synthesis of selenium-labeled proteins. The second regulation is to add 22.5 µM selenocysteine and 5 µM methionine as sulfur / selenium-containing amino acid precursors required for the synthesis of target proteins when the bacteria grow to the induction stage. Six hours after induction, the same proportion of 22.5 µM selenocysteine and 5 µM methionine is again supplemented. The above supplementing method follows the three stages of high-sulfur maintenance, low-sulfur adaptation, and induction supplementation.

[0067] Compared with Example 2, in this embodiment, selenocysteine (selenocysteine is the oxidized form of selenium cysteine, which exists stably in air and can be reduced to selenium cysteine in solution) and methionine are directly added at the second regulation to ensure sufficient synthesis of target proteins, while also meeting the selectivity of labeling sites and improving the cysteine labeling efficiency to 86% per site. This can avoid the preferential metabolism of selenium into selenomethionine when selenate is added, which is then absorbed by the protein. Under high-selenium conditions, selenomethionine SeMet does not convert into selenocysteine SeCys in large quantities, resulting in labeling on methionine. Therefore, by directly adding selenocysteine and methionine in this embodiment, the low cysteine labeling efficiency can be avoided, which affects the efficient and specific labeling of selenium cytochrome c.

[0068] (5) Specific process of expression and purification of selenium-labeled proteins

[0069] Protein expression:

[0070] Take a prepared competent cell (stored in a -80°C refrigerator) and add a plasmid with a final concentration of 100-150 ng / µL. Gently tap the bottom of the test tube to mix the contents. After placing the Ep tube in a 42°C constant temperature water bath for 90 s and on ice for 1 min, add 500 µL of LB medium (without antibiotics) and incubate at 37°C, 220 rpm on a shaker for 30-60 min. After centrifugation, resuspend with 100 µL of medium, then plate (ampicillin resistance), and invert in a 37°C incubator overnight to allow the cells to form colonies on the plate.

[0071] The next day, liquid medium (Table 1, Table 2) was prepared, 20 mL of the medium was taken into a small conical flask, and a single colony was picked from the plate in the above step and inoculated into 20 mL of the medium in a small conical flask. At the beginning of the bacterial culture, 5 mM Na2SO4was supplemented as a sufficient sulfur source, and the culture was incubated for about 13-15 h, and the OD was measured 600 When the OD was 1-1.5, 1 mL of the culture was transferred into 1 L of the medium in a large bottle, and the culture was incubated overnight and supplemented with 50 μM Na2SO4. This was to enable the bacteria to adapt to a low-sulfur environment and establish a sulfur-starved state.

[0072] On the third day, when the bacteria grew to the induction stage, the OD was measured 600 When the OD was about 0.75, 22.5 μM selenocystine and 5 μM methionine were added as sulfur / selenium amino acid precursors required for the synthesis of target proteins. The culture was maintained at 37°C, 220 rpm, for 1 h, and then 1 mM IPTG was added, and the culture was incubated at 30°C, 70 rpm, for 6 h. Then, 22.5 μM selenocystine and 5 μM methionine were added again, and the culture was incubated at 30°C overnight.

[0073] On the fourth day, the bacteria were collected by centrifugation at 6000 rpm for 15 min, and the bacteria were stored at -20°C or directly subjected to the next step of protein extraction and purification.

[0074] Protein purification:

[0075] The bacteria were collected by centrifugation (8000 rpm, 10 min) to remove the culture medium, and the collected bacteria 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 bacteria)) and 5 μl of DNA digestion enzyme (DNase I) were added and digested for 1 h. Then, the bacteria were fully broken by high pressure, and then centrifuged at 20000 rpm for 30 min. 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 h, and then centrifuged at 20000 rpm for 30 min. The supernatant was dialyzed overnight (5 L: Na2HPO4 8.66 g, NaH2PO4 4.68 g, pH 7.0).

[0076] The liquid after overnight dialysis was centrifuged at 20000 rpm for 30 min, and the supernatant was purified by SP cation column and molecular sieve Pg75, and the cytochrome c component was collected.

[0077] In this step, the SP cation column is passed through, and the buffer A and B used are: A buffer-20 mM PB (pH=7.0), B buffer-20 mM PB and 1M NaCl (pH=7.0); the buffer for passing through the molecular sieve Pg75 is 20 mM PB, 150 mM NaCl (pH=7.0).

[0078] 4. Verification method of labeling efficiency: ESI-MS technology is used to characterize the selenium-labeled protein. The mobile phase is selected as an acetonitrile-water system (50% acetonitrile when the target protein elutes).

[0079] 5. Control group setting: set the unlabeled cytochrome c as the control group, and the treatment conditions are consistent with the experimental group.

[0080] II. Experimental results:

[0081] The obtained selenium cytochrome c sample is identified by a bottom-up secondary mass spectrometry strategy. That is, the extracted protein in the sample is subjected to reduction, alkylation and enzymolysis steps to become a peptide mixture, and the peptide is then scanned by electrospray into mass spectrometry to obtain mass spectrometry spectrum, and finally the relevant information of the protein in the sample can be obtained by analyzing these mass spectrometry spectrum through related software.

[0082] Specific analysis as follows: the mass spectrometry raw data is analyzed by MaxQuant (2.2.0.0) software, and the database search algorithm used is the Andromeda built-in software. The database used for searching is the Escherichia coli protein sequence database downloaded from Uniprot. The main search parameters are set as default, and the key parameters are described as follows: variable modification is set as Carbamidomethyl(C)-57.021464Da, Oxidation(M)-15.994915, Acetyl(ProteinN-term)-42.010565Da, Se substitution S-47.944451Da, and fixed modification is set as Carbamidomethyl(C)-57.021464Da; the enzyme cutting condition is set as Trypsin / P, with a maximum of 2 missed cutting sites; the MS1 mass tolerance of firstsearch and mainsearch is set as 20ppm and 10ppm respectively; the MS2 mass tolerance is set as 20ppm. Proteins that cannot be distinguished by unique peptides are grouped into the same protein group by MaxQuant software. The search results are filtered by 1% FDR (protein and peptide level).

[0083] The results show that the single cysteine selenium-labeled protein accounts for 46%, the double cysteine selenium-labeled protein accounts for 40.1%, and the non-selenium-labeled protein accounts for 13.7% in the yeast-derived cytochrome c sample obtained by the embodiment Figure 1 ). The double selenium-labeled site is accurately located in the cytochrome c by secondary mass spectrometry verification, which is coordinated with the heme iron of the cysteine residues (cys14, cys17), the highest single residue labeling efficiency reaches 86%, and the target protein yield is stably at 1 mg / g (protein / wet mycelium).

[0084] As shown in Figure 5 , 50 mM selenium-labeled yeast-derived cytochrome c and 50 mM non-labeled yeast-derived cytochrome c prepared in the embodiment are respectively dissolved in 50 mM phosphate buffer and 100 mM potassium ferricyanide to prepare an oxidation state (OX), and 50 mM selenium-labeled yeast-derived cytochrome c and 50 mM non-labeled yeast-derived cytochrome c prepared in the embodiment are respectively dissolved in 50 mM phosphate buffer and 100 mM ascorbic acid to prepare a reduction state (re), which are respectively detected on a circular dichroism instrument. The results show that there is almost no difference between the selenium-labeled cytochrome c prepared in the embodiment and the wild-type cytochrome c in terms of oxidation state or reduction state structure, which indicates that the method of the embodiment can be used for biomolecular structure research.

[0085] As shown in Figure 7 , the one-dimensional 77 Se NMR spectrum of the selenium cysteine cytochrome c prepared in the embodiment at a concentration of 1.5 mM, with a sampling time of 9h30min, can clearly show the signal of selenium atoms.

[0086] As shown in Figure 8 , the two-dimensional 1 H- 77 Se HSQC correlation spectrum of the selenium cysteine cytochrome c prepared in the embodiment in the oxidation state and the reduction state, which indicates that the selenium atoms introduced in the protein of the embodiment can be used as a probe to react with the conformational change of the protein.

[0087] The above experimental results show that the specific selenium labeling of the cysteine residues in cytochrome c is successfully realized by the selenium labeling technology of the embodiment, the unit point labeling efficiency reaches 86%, and the protein yield is increased to 1 mg / g (protein / wet mycelium). The conformation of the labeled protein is basically unchanged, which provides a high-fidelity probe for the conformational study of the active site of the protein centered on the heme. The traditional selenium-sulfur inorganic salt replacement method of embodiment 2 has significant non-specific labeling (the methionine site accounts for >70%, Figure 3 ), and the labeling efficiency of the 20 kinds of amino acid replacement method of embodiment 3 is less than 40% (LC-MS / MS verification, Figure 4 ).

[0088] Example 2: Selenium cytochrome c expressed by selenium-sulfur inorganic salt substitution method

[0089] I. Experimental materials and methods:

[0090] 1. According to the literature reported selenium-sulfur inorganic salt addition method, the specific steps of selenium cytochrome c expression and purification are as follows:

[0091] Protein expression:

[0092] Take a prepared BL21 competent cell (stored in -80°C refrigerator), add the same yeast-derived cytochrome c recombinant plasmid as in Example 1, the final concentration of the plasmid is 100-150 ng / μL, gently hit the bottom of the test tube to mix the contents; after placing on ice for 30 min, put the Ep tube into a 42°C constant temperature water bath for 90 s, and place on ice for 1 min; add 500 μL of LB medium (without antibiotics), 37°C, 220 rpm shaking bed culture for 30-60 min; after centrifugation, resuspend with 100 μL of medium, then plate (ampicillin resistance), and invert in a 37°C incubator overnight culture to allow the cells to form colonies on the plate.

[0093] The next day, prepare the liquid medium (Table 1), take 20 mL of the medium into a small conical flask, and pick a single colony from the plate in the above step into the 20 ml small conical flask. In the early stage of bacterial culture, supplement 5 mM Na2SO4 as an adequate sulfur source, and culture for about 13-15 h. When the OD 600 is 1-1.5, take 1 ml of the culture and transfer it into 1 L of medium in a large bottle, and incubate overnight and supplement 50 μM Na2SO4 to promote the adaptation of the bacteria to a low-sulfur environment and establish a sulfur-starved state.

[0094] On the third day, when the bacteria grow to the induction stage, measure the OD 600 is about 0.75, add a specific ratio of sodium selenite and sodium sulfate, maintain 37°C, 220 rpm, after 1 h, add 1 mM of inducer, and culture at 30°C, 70 rpm for 6 h, then again supplement the same ratio of sodium selenite and sodium sulfate, and shake culture overnight at 30°C.

[0095] On the fourth day, centrifuge at 6000 rpm for 15 min to collect the bacterial cells, and store at -20°C, or directly proceed to the next step of protein extraction and purification.

[0096] Protein purification:

[0097] Collect the bacteria, remove the culture medium by centrifugation (8000 rpm, 10 minutes), resuspend the collected bacteria in 40 mL lysis buffer (50 mM Tris-HCl (6.057 g / L), 5 mM EDTA-2Na (1.86 g / L), pH 7.5), add lysozyme (Eggwhite, 1.5 mg / g (lysozyme / wet bacteria)) and 5 μl DNA digestion enzyme (DNase I) for 1 h, then add high pressure to fully break the bacteria, and then centrifuge at 20000 rpm for 30 min; place the supernatant after centrifugation in a clean 250 ml beaker, slowly add ammonium sulfate (150 g / L) for salting out for 3-6 h, and then centrifuge at 20000 rpm for 30 min, and then dialyze the supernatant overnight (5 L: Na2HPO4 8.66 g, NaH2PO4 4.68 g, pH 7.0).

[0098] Centrifuge the liquid after overnight dialysis at 20000 rpm for 30 min, and purify the supernatant with SP cation column and molecular sieve Pg75 in sequence to collect the cytochrome c component.

[0099] In this step, the SP cation column is passed through, and the buffer A and B used are: A buffer-20 mM PB (pH=7.0), B buffer-20 mM PB and 1 M NaCl (pH=7.0); the buffer for passing through the molecular sieve Pg75 is 20 mM PB, 150 mM NaCl (pH=7.0).

[0100] 2. Verification method of labeling efficiency: ESI-MS technology is used to characterize the selenium-labeled protein. The mobile phase is acetonitrile-water system (acetonitrile accounts for 50% of the target protein elution).

[0101] 3. Control group setting: set the unlabeled cytochrome c as the control group, and the treatment conditions are consistent with the experimental group.

[0102] II. Experimental results

[0103] In this experiment, two groups of proportions were made to observe the labeling efficiency, such as Figure 2 , wherein the selenium-sulfur inorganic salt ratio is 0.5 (i.e. 1:1), which means that the sodium selenite and sodium sulfate added in the culture medium are both 25 μM, and the selenium-sulfur inorganic salt ratio is 0.9, which means that the sodium selenite and sodium sulfate added in the culture medium are 22.5 μM and 5 μM respectively. Mass spectrometric analysis found that when the selenium-sulfur inorganic salt ratio was 0.9 (i.e. 9:1), the primary mass spectrum showed high labeling efficiency.

[0104] Therefore, the yeast-derived cytochrome c expressed by the 0.9 proportion selenium-sulfur inorganic salt was subjected to secondary mass spectrometry for selenium labeling site identification. The method was the same as described in Example 1. The final analysis result was that the selenium labeling site accounted for 72.83% at the methionine No. 80 position of the protein and 9.68% at the methionine No. 64 position of the protein (Figure 3). The cysteine No. 14 and 17 positions stably connected with the heme in the active site were not labeled by selenium.

[0105] Example 3: Selenium cytochrome c expressed by 20 amino acids respectively added method

[0106] I. Experimental materials and methods:

[0107] 1. The supplementing method of sulfur / selenium source: According to the literature report, the main difference between this method and the selenium-sulfur inorganic salt adding method is that the sulfur source is not strictly limited in the culture medium, and the selenium labeling is mainly achieved by supplementing a large amount of selenocysteine and other amino acids required for protein synthesis in the protein expression stage, so that the protein is labeled with selenium during synthesis.

[0108] 2. The specific experimental steps are as follows:

[0109] Protein expression:

[0110] Take a prepared BL21 competent cell (stored in a -80°C refrigerator), add the same yeast-derived cytochrome c recombinant plasmid as in Example 1, and the final concentration of the plasmid is 100-150 ng / μL. Gently tap the bottom of the test tube to mix the contents evenly; after placing the Ep tube in a 42°C constant temperature water bath for 90 s on ice for 1 min; add 500 μL of LB medium (without antibiotics), 37°C, 220 rpm shaking bed culture for 30-60 min; after centrifugation, resuspend with 100 μL of medium, then plate (ampicillin resistance), and invert in a 37°C constant temperature incubator for overnight culture to allow the cells to form colonies on the plate.

[0111] The next day, pick a single clone in 1 ml LB and culture at 37°C, 220 rpm shaking bed for about 12 h.

[0112] 3. Configure the medium (stage one) by 25 mM Na2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, 5 mM Na2SO4 in a 1 L conical flask, high temperature sterilization, and then add MgSO4, trace metal mixture, vitamin B 12 , vitamin mixture (as shown in Table 2), 10 mM glucose, ampicillin, and heme precursor for synthesizing heme prosthetic group. Take 100 ml of the medium in a small conical flask and culture for 4-6 hours, then transfer to 1 L of medium and culture to OD 600=1-1.5, the ingredients in the second stage were added, consisting of 10 mM glucose, aspartate and 17 amino acids and methionine with selenocysteine. At 37 o C, 220 rpm for 10 min, then changed to 30 o C, 70 rpm, after 10 min recovery, IPTG 1 mM was added to start induction, and expressed overnight.

[0113] The medium formula here is described in Table 3 below:

[0114] Table 3 Medium formula

[0115]

[0116] The formula of the trace metal mixture in the above table is shown in Table 4 below:

[0117] Table 4 Formula of trace metal mixture

[0118]

[0119] On the third day, centrifugation was performed at 6000 rpm for 15 min, the supernatant was discarded, and the bacterial cells were collected and stored at -20°C, or directly subjected to the next step of protein extraction and purification.

[0120] The subsequent protein purification steps were the same as in Example 1 and Example 2.

[0121] II. Experimental results

[0122] This experiment tried two groups of induction expression conditions, respectively at OD 600 =1 and 1.5 to add amino acids. As Figure 4 shown, verified by mass spectrometry, the final protein selenium labeling efficiency of the two groups of experiments was very low. The expression group with OD 600 =1.5 to add amino acids, the final labeling efficiency was slightly higher, but still less than 40%.

[0123] Example 4 Selenium labeling of active proteins containing free sulfhydryl (-SH)

[0124] Ubiquitin (Ub) is a highly conserved eukaryotic regulatory protein (76 amino acids, 8.451 kDa) that regulates protein degradation, cell cycle, signal transduction and other biological processes through ubiquitination modification. Its core function is achieved through the ubiquitination modification pathway, and the polyubiquitin chain at K48, K63 and K11 is recognized by 26S proteasome. The ubiquitin receptor (such as UBR1) of the regulatory subunit (19S) specifically binds the ubiquitin chain, and drives the 20S core subunit to hydrolyze the substrate under the energy supply of ATPase (ΔG = -7.3 kcal / mol). Monoubiquitination modification can mediate membrane protein endocytosis and vesicle transport (such as the EGFR internalization process). Studies have shown that the ubiquitin mutant with K63 site mutated to cysteine (K63C) can block the formation of polyubiquitin chain (K48 / K63 heterotypic connection efficiency decreased by >95%), providing an ideal model for the study of monoubiquitination dynamic process.

[0125] In this embodiment, the selenium labeling technology of the application is used for specific selenium labeling of the mutation site of the ubiquitin K63C mutant, so that 77 The high sensitivity of Se NMR is used to study the structure-activity relationship of ubiquitin.

[0126] I. Experimental materials and methods

[0127] 1. Source of ubiquitin K63C mutant: the K63 site of wild-type ubiquitin is mutated to cysteine by site-directed mutagenesis to construct the ubiquitin K63C mutant. The ubiquitin K63C mutant is expressed in E. coli, and IPTG is used to induce expression, and the culture is incubated at 37°C for 4 hours. The protein is purified by affinity chromatography and gel filtration.

[0128] 2. Expression and purification steps of selenium-labeled ubiquitin K63C mutant:

[0129] Protein expression:

[0130] Take a prepared BL21 competent cell (stored in a -80°C refrigerator), add the constructed ubiquitin K63C mutant plasmid, and the final concentration of the plasmid is 100-150 ng / μL. Gently tap the bottom of the test tube to mix the contents evenly. After placing the Ep tube in a 42°C constant temperature water bath for 90s and placing it on ice for 1 min, add 500 μL of LB medium (without antibiotics), and incubate at 37°C, 220 rpm on a shaker for 30-60 min. After centrifugation, resuspend with 100 μL of medium, then plate (ampicillin resistance), and invert in a 37°C incubator overnight to allow the cells to form colonies on the plate.

[0131] The next day, the liquid medium (Table 1, Table 2) is configured, 20 mL of the medium is taken into a small conical flask, and a single colony is picked from the plate in the above step and placed in the 20 ml small conical flask. In the early stage of bacterial culture, 5 mM Na2SO4 is supplemented as a sufficient sulfur source, and the culture is cultured for about 13-15 h, and the OD is measured 600 When the OD is 1-1.5, 1 ml of the culture is transferred into 1 L of the medium in a large bottle, and the culture is cultured overnight and supplemented with 50 μM Na2SO4 to adapt the bacteria to a low-sulfur environment and establish a sulfur starvation state.

[0132] On the third day, when the bacteria grow to the induction stage, the OD is measured 600 When the OD is about 0.75, 22.5 μM selenocystine and 5 μM methionine are added as sulfur / selenium amino acid precursors required for synthesis of the target protein, and the culture is maintained at 37°C, 220 rpm, for 1 h, then 1 mM IPTG is added, and the culture is cultured at 37°C for 4 h, then 22.5 μM selenocystine and 5 μM methionine are added again, and the culture is cultured at 37°C for 4 h, and then the bacteria are collected. The bacteria are collected by centrifugation at 6000 rpm for 15 min, and are stored at -20°C, or are directly subjected to the next step of protein extraction and purification.

[0133] Protein purification:

[0134] The collected bacteria are resuspended in 20 mM sodium acetate (pH=5.0), 3 mM DTT, and are subjected to high-pressure crushing, and then are centrifuged at 20000 rpm for 30 min. The supernatant after centrifugation is placed in a clean 250 ml beaker, and is subjected to separation and purification by an SP cation column. The buffer used is buffer A-20 mM sodium acetate, 3 mM DTT, pH=5.0; and buffer B-20 mM sodium acetate, 1 M NaCl, 3 mM DTT, pH=5.0. Gradient separation is used, and the target protein is eluted at 30% buffer B. The target protein component is collected and concentrated to 4 mL, and then is further purified by molecular sieve Pg75, and the buffer used is the above buffer A, and the target protein component is eluted at 60 mL. The target protein is collected and stored at 4 o C Short-term storage.

[0135] 3. Control group setting: The unlabelled ubiquitin K63C mutant is set as a control group, and the treatment conditions are consistent with those of the experimental group.

[0136] II. Experimental results:

[0137] After mass spectrometry identification, the specific selenium labeling of the ubiquitin K63C mutant at the 63rd cysteine is successfully realized by using the selenium labeling method of the application, and the labeling efficiency reaches 64%. As shown in FIG. 2, the mass spectrometry results show that the selenium atom is successfully incorporated into the 63rd cysteine of the ubiquitin K63C mutant. Figure 6As shown, 8540 Da is the non-labeled molecular weight of the protein, 8600 Da is the molecular weight of the protein after selenium labeling, and there is an increase of 60 Da after selenium labeling due to the binding of a methyl group at the corresponding site (a simple selenium labeling increases by 46 Da).

Claims

1. A method for efficient selenium labeling of cysteine-containing proteins, comprising: The plasmid expressing the target protein is introduced into an E. coli expression system, and the ratio of selenium and sulfur elements in the nutrition related to protein synthesis in the culture medium is regulated twice during the bacterial culture process, so that the target protein is expressed and labeled in the E. coli expression system; the first regulation includes establishing a sufficient sulfur source environment in the early stage of bacterial growth, and then gradually reducing the sulfur source concentration to induce a sulfur starvation state; The second regulation includes supplementing methionine and selenocysteine related to the synthesis of the target protein into the culture medium twice, and the ratio of selenocysteine to methionine is 1-9:

1.

2. The method of claim 1, wherein the efficient selenium labeling of cysteine-containing proteins is achieved by, The target protein is a biological macromolecule formed by linking amino acids by peptide bonds, including peptide segments, proteins and peptide complexes.

3. The method for achieving efficient selenium labeling of cysteine-containing proteins according to claim 1, characterized in that, The target protein is cytochrome c.

4. The method for achieving efficient selenium labeling of cysteine-containing proteins according to claim 3, characterized in that, In the second regulation, the molar concentration ratio of selenocysteine and methionine added into the culture medium in the first and second times is 4.5:

1.

5. The method for achieving efficient selenium labeling of cysteine-containing proteins according to claim 4, characterized in that, The preparation of the selenium-labeled cytochrome c includes the following steps, (1) Expression of the selenium-labeled target protein: First, the plasmid of the target protein is introduced into E. coli, and the introduced E. coli is uniformly coated on LB solid medium and cultured in a 37°C incubator overnight; The next day, liquid medium was prepared and 20 ml of this medium was placed in a small conical flask. A single colony from the plate of the previous step was picked and placed in the 20 ml of medium in the small conical flask and supplemented with 5 mM Na2SO4. The culture was incubated for 13-15 h and the OD was measured 600 When the OD was between 1 and 1.5, 1 ml was transferred to 1 L of overnight culture medium and supplemented with 50 μM Na2SO4. Day 3, measure OD 600 When OD is about 0.75, add the first 22.5 μM selenocystine and 5 μM methionine, keep 37°C, 220 rpm, 1 h, then add inducer 1 mM, keep 30°C, 70 rpm, 6 h, then add the same proportion of selenocystine and methionine again; The fourth day of overnight culture, centrifuge the bacteria at 6000 rpm for 15 min, and store at -20°C or directly proceed to the next step of protein extraction and purification; (2) Purification method of selenium-labeled cytochrome c: Take the bacteria just centrifuged or stored at -20°C, resuspend in 40 mL lysis buffer, the composition of lysis buffer is: 50 mM Tris-HCl, 5 mM EDTA-2Na, pH 7.5, add lysozyme and DNA nuclease for 1 h, then add high pressure to fully break the bacteria, then centrifuge at 20000 rpm for 30 min; The supernatant after centrifugation is placed in a clean 250 ml beaker, 150 g / L ammonium sulfate is slowly added for 3-6 h, and then centrifuged at 20000 rpm for 30 min, and the supernatant is dialyzed overnight; The liquid after overnight dialysis is centrifuged at 20000 rpm for 30 min, and the supernatant is purified by SP cation column and molecular sieve Pg75 in turn, and the cytochrome c component is collected, wherein the lysozyme is added at a ratio of 1.5 mg / g=lysozyme / wet bacteria, and the DNA nuclease is added at a ratio of DNase I / bacterial liquid volume=1 μl / ml.

6. The method of claim 5, wherein the cysteine-containing protein is efficiently labeled with selenium. The preparation method of the liquid medium in step (1) is: 50 mM Na2HPO4, 50 mM KH2PO4, 50 mM NH4Cl, 10 mM NaCl are configured in a 1L conical flask, autoclaved, and then 2 mM MgCl2, trace metal mixture, vitamin mixture, 0.4% glucose, 200 μM calcium chloride, 100 μg / mL ampicillin antibiotic and precursors for synthesizing heme cofactors are added. ​ 7. The method of claim 5, wherein the cysteine-containing protein is efficiently labeled with selenium. The buffer A and B used in the step (2) are as follows: the buffer A is 20 mM PB, the buffer B is 20 mM PB and 1 M NaCl, the pH of the buffer A and B is 7.0, the buffer used in the step of passing through the molecular sieve Pg75 is 20 mM PB and 150 mM NaCl, and the pH of the buffer used in the step of passing through the molecular sieve Pg75 is 7.

0. ​

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