Method for crystallizing BM3 protein in bacillus megatherium

Abstract

The invention discloses a method for crystallizing BM3 protein in bacillus megaterium. The method comprises the following steps: constructing a gene for coding the BM3 protein into an expression vector, carrying out induced expression in escherichia coli, carrying out affinity chromatography purification, excising a fusion tag, carrying out gel filtration purification, incubating with cofactors FAD, FMN and NADPH to form a compound, and crystallizing under a specific pool liquid condition through a seated drop gas phase diffusion method. According to the method, the high-quality BM3 protein crystal suitable for X-ray diffraction analysis can be obtained, and a basis is provided for analyzing the three-dimensional structure of the BM3 protein crystal and researching an electron transfer mechanism.

Description

Technical Field

[0001] This invention belongs to the field of structural biology technology, specifically relating to a method for obtaining BM3 protein crystals in an expression system and the BM3 protein crystals prepared by the method, which is applicable to the analysis of protein three-dimensional structure and the study of functional mechanisms. Background Technology

[0002] The functional mechanisms of proteins are hidden within their three-dimensional structures. Determining protein structures helps to understand their functional mechanisms through the interactions between biomolecules, thereby solving some biological problems. X-ray crystallography is a primary method for analyzing protein structures. X-rays are high-energy, short-wavelength electromagnetic waves. When X-rays strike molecular crystal particles, they interact with electrons in the crystal, producing a diffraction effect. By collecting these diffraction signals with a detector, the distribution of electron density in the crystal can be determined, thus obtaining information about the particle positions and providing a basis for analyzing the crystal structures of biomolecules. After protein crystallization, its three-dimensional structure can be seen through X-ray diffraction. However, this method cannot be used to analyze proteins with large molecular weights, and it has high requirements for protein crystallization, thus limiting its application to the structural analysis of proteins that cannot be crystallized.

[0003] Cytochrome P450 is a superfamily of proteases containing type b heme, widely distributed in organisms in nature. It is one of the oldest and largest enzyme families, named for the maximum absorption peak near 450 nm of the complex formed when the reduced state binds to CO. BM3 is a classic self-sufficient monooxygenase within cytochrome P450. It is the third P450 enzyme discovered in *Bacillus megaterium*, hence its name. It is a single-stranded, multi-domain enzyme with high stability, high catalytic activity, and a molecular weight of approximately 119 kDa. BM3 protein, also known as CYP102A1, is a large motor-like fusion oxidase containing a heme-binding domain, a flavin-binding electron transport domain, a heme domain, and a reductase domain similar to mammalian CPR. Its unique structure-function relationship in substrate oxidation and electron transport has made it a hot research topic. However, its large molecular weight and structural complexity make obtaining high-quality single crystals difficult.

[0004] Existing techniques have reported crystal structures of different domains of BM3 and structural information on several truncated variants, but high-resolution crystal structures of full-length BM3 or cofactor complexes in specific states are scarce. Conventional crystallization strategies include truncating flexible fragments, introducing mutations to promote lattice contacts, adding crystallization chaperones and cofactors to stabilize conformations. Cryo-electron microscopy is also used to resolve protein structures, but this method relies on sample homogeneity and stability; conformational heterogeneity of proteins in solution reduces the final resolution.

[0005] There is an urgent need in this field for a high-quality protein crystallization method to resolve the structure of BM3 protein in Bacillus megaterium. Developing a stable, reproducible expression, purification, and crystallization process suitable for obtaining diffractive crystals is of great significance. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing BM3 protein in an expression system and obtaining crystals that can be used for X-ray diffraction, so as to provide a structural basis for resolving the full-length three-dimensional structure of BM3 and studying its electron transport mechanism, and to provide BM3 protein crystals prepared by this method as protected objects.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for crystallizing BM3 protein from Bacillus megaterium includes the following steps:

[0009] 1) The gene encoding the BM3 protein was constructed into a vector suitable for expression in E. coli and amplified in cells to obtain an expression plasmid;

[0010] 2) The obtained expression plasmid was transformed into an E. coli host and cultured in liquid medium until the cell density OD reached 100%. 600 The concentration was 0.6-0.8, and then the culture temperature was lowered to 23℃ and expression was induced with isopropyl-β-D-thiogalactoside at a final concentration of 0.4 mM. At the same time, 5-aminolevulinic acid and riboflavin, both at a final concentration of 0.4 mM, were added to the induction culture system and cultured with shaking at 160 rpm for 40 hours.

[0011] 3) After cell harvesting, the recombinant BM3 protein with the fusion tag was purified by affinity chromatography, and the fusion tag was removed by a specific protease, followed by gel filtration and purification to a monodisperse state.

[0012] 4) The purified BM3 protein, flavin cofactors FAD and FMN, and reduced nicotinamide adenine dinucleotide NADPH were incubated in a suitable buffer at 4°C for 2 h in the dark to obtain the cofactor-bound protein complex.

[0013] 5) Crystallization was carried out using the sitting drop gas phase diffusion method at a crystallization temperature of 18℃, a pool liquid volume of 200 μL, and a drop volume of 1 μL protein solution plus 1 μL pool liquid. The pool liquid conditions for the growth of BM3 protein crystals after initial screening were 0.01 M nickel chloride, 0.1 M Tris buffer and 20% PEG 2000 MME.

[0014] The method can obtain BM3 protein crystals suitable for X-ray diffraction analysis;

[0015] Preferably, the expression vector contains an affinity tag to facilitate affinity chromatography purification and includes a protease cleavage site for removing the affinity tag.

[0016] Preferably, the binding buffer used in the affinity chromatography contains 20 mM Tris-HCl, pH=7.5, 150 mM sodium chloride and 5 mM imidazole, and the elution buffer contains 200 mM imidazole.

[0017] Preferably, the protease used for protease digestion is TEV protease and the protease digestion is performed at 4-16°C, with a molar ratio of protease to target protein of 1:10-100.

[0018] Preferably, the purified protein is concentrated to 5-20 mg / ml for crystallization screening.

[0019] Preferably, the buffer solution is 20 mM Tris-HCl, pH=7.5, and 150 mM sodium chloride.

[0020] Preferably, a reducing agent of 1-5 mmol / L is added to the reaction system before the co-incubation step to keep the cofactor in a reduced or controlled oxidation state.

[0021] Preferably, the crystallization employs micro-seedling to promote crystal growth.

[0022] The present invention also provides a BM3 protein crystal, which is prepared by the protein crystallization method described above.

[0023]

[0024] BM3 is 1049 AA in length, and its amino acid sequence is shown in SEQ ID NO.2:

[0025]

[0026] Excellently, the crystal exhibits diffraction capability in X-ray diffraction experiments that can be used for three-dimensional structure analysis.

[0027] The beneficial effects of this invention are:

[0028] The above-mentioned technical solution enables the crystallization preparation of BM3 protein complexes containing cofactor binding. This method, through the synergistic effect of host selection, optimization of induced expression parameters, tag-assisted purification and enzymatic removal, co-incubation of cofactors to stabilize conformation, and a gas-phase diffusion crystallization strategy under specific solution conditions, is conducive to improving the crystallization success rate and obtaining crystals suitable for X-ray diffraction. This provides a reliable experimental basis for resolving the full-length three-dimensional structure of BM3 and studying its electron transport mechanism. Attached Figure Description

[0029] Figure 1 This is a preliminary crystal image of BM3;

[0030] Figure 2 Agarose gel electrophoresis image of plasmids successfully cloned from the BM3 molecule, verified by double enzyme digestion.

[0031] Figure 3 SDS-PAGE image of Ni-NTA purified BM3 protein with MBP tag;

[0032] Figure 4 SDS-PAGE image of BM3 protein after the MBP tag was removed by TEV enzyme;

[0033] Figure 5 Gel filtration chromatography and SDS-PAGE image of BM3 protein at half maximum (HHM).

[0034] Figure 6 A schematic diagram of the crystals after adding the additives;

[0035] Figure 7 A schematic diagram of BM3 crystals incubated with FAD, FMN, NADPH, and arachidonic acid. Detailed Implementation

[0036] The present invention will be further described below through specific embodiments. To make the inventive objectives, technical solutions, and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the embodiments described in this specification are merely for explaining the present invention and are not intended to limit the present invention.

[0037] Unless otherwise stated, all films and reagents used in the examples are commercially available or synthesized using conventional methods and can be used directly without further processing, as are the instruments used in the examples.

[0038] Example 1:

[0039] (1) Construction of the recombinant expression vector for BM3 protein: The BM3 gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the selected vector was pCW-E-HTH-3. Nde I and Xho I were selected as restriction sites using Snapgene software, and appropriate primers were designed to amplify the BM3 gene by PCR to obtain the target fragment. After double digestion of the target fragment and the vector, ligation was performed using T4 DNA rapid ligase. The ligation system was then transformed into DH5α competent cells, plated, and incubated overnight at 37°C. The next day, single clones were picked and incubated overnight at 37°C using LB medium at 220 rpm. Recombinant plasmids were then extracted. Double digestion identification was then performed, and the results were as follows: Figure 2 As shown, sample number three was sent for sequencing, and the results were consistent.

[0040] (2) BM3 protein expression: The recombinant plasmid with correct sequencing in step (1) was transformed into Rosetta 2 (DE3) competent cells, plated, and incubated overnight at 37°C. The next day, single clones were picked and incubated overnight at 37°C at 220 rpm for preservation. The bacterial culture was then inoculated into 1 L TB culture medium and cultured at 37°C with shaking at 220 rpm until the OD600 reached 0.6~0.8. The culture was then cooled to 23°C, and IPTG, 5-ALA, and Riboflavin were added to a final concentration of 0.4 mM. The culture was then incubated at 23°C with shaking at 160 rpm for 40 h, and the bacterial cells were collected for subsequent use.

[0041] (3) BM3 protein purification: The bacterial cells collected in step (2) were resuspended in a buffer of 20 mM Tris-HCl pH 7.5, 150 mM NaCl, and 5 mM imidazole, and then sonicated. The supernatant was collected by centrifugation at 15,000 rpm for 30 min at 4°C. The supernatant was then incubated with nickel gel equilibrated with the same buffer used during the disruption at 4°C for 1 h. The incubated protein-nickel gel mixture was then transferred into a Bio-Rad gravity column for flow-through (Ft). Subsequently, different concentration gradients of imidazole buffers were used for the following steps: washing of contaminants 1 (5E; 5 mM IM+ buffer), washing of contaminants 2 (10E; 10 mM IM+ buffer), elution of the target protein (200E; 200 mM IM+ buffer), and final elution (500E; 500 mM IM+ buffer). Samples were taken from the entire Ni-NTA purification process and analyzed by SDS-PAGE. The results are as follows: Figure 3 As shown. The eluted target protein was mixed with TEV enzyme at a molar ratio of protein:TEV enzyme = 10:1, and incubated overnight at 4°C by rotation. The next day, the protein was reverse-coated onto a nickel column, and the SDS-PAGE results are shown below. Figure 4 As shown. After collecting the target protein, gel filtration chromatography was performed, and the sample at the half-peak was used for subsequent experiments. The gel filtration chromatography diagram and the SDS-PAGE results of the protein at the half-peak are shown below. Figure 5 As shown, the target protein with high purity and good uniformity was obtained.

[0042] (4) Crystallization of BM3 protein after incubation with FAD, FMN, and NADPH: The target protein obtained in step (3) was incubated with FAD, FMN, and NADPH for 2 hours, with a molar ratio of BM3:FAD:FMN:NADPH = 1:5:5:10. Then, the BM3 protein incubated with the above small molecules was subjected to initial crystal screening using a gas-phase diffusion method. Specifically, 200 μL of pooling solution was added to each well of a 48-well crystal plate, and 1 μL of protein and 1 μL of pooling solution of different concentrations were added to both sides of the pooling solution. The crystal plate was sealed and crystals were grown at 18°C. Finally, BM3 initial screening crystals were obtained under the conditions of 0.01M Nickel chloride, 0.1M Tris, and 20% PEG 2000 MME, as shown in the figure. Figure 1 As shown.

[0043] Comparative Example 1: BM3 protein does not incubate cofactor sieve crystals

[0044] The target protein obtained in step (3) of Example 1 was directly screened for crystal formation by gas-phase diffusion at concentrations of 5 mg / mL and 10 mg / mL. Specifically, 200 μL of pooling solution was added to each well of a 48-well crystal plate, and 1 μL of protein and 1 μL of pooling solution at different concentrations were added to both sides of the pooling solution. The crystal plate was then sealed and crystals were grown at 18°C. No crystals grew.

[0045] Comparative Example 2: BM3 protein was incubated with FAD and FMN and then screened into crystals.

[0046] The target protein obtained in step (3) of Example 1 was co-incubated with FAD and FMN for 2 hours, with a molar ratio of BM3:FAD:FMN = 1:5:5. Afterwards, the concentration of BM3 protein incubated with the above small molecules was adjusted to 5 mg / mL and 10 mg / mL, and then subjected to initial crystal screening by gas-phase diffusion. Specifically, 200 μL of pooling solution was added to each well of a 48-well crystal plate, and 1 μL of protein and 1 μL of pooling solution of different concentrations were added to both sides of the pooling solution. The crystal plate was then sealed and crystals were grown at 18°C. No crystal growth was observed.

[0047] Comparative Example 3: After incubating BM3 protein with FAD, FMN, and NADPH, an additive was added to screen the crystals.

[0048] The target protein obtained by the same procedure as in step (3) of Example 1 was incubated with FAD, FMN, and NADPH for 2 hours, with a molar ratio of BM3:FAD:FMN:NADPH = 1:5:5:10. Afterwards, the concentrations of the incubated BM3 protein were adjusted to 5 mg / mL and 10 mg / mL, and then initial crystal screening was performed using a gas-phase diffusion method. Specifically, 200 μL of pooling solution was added to each well of a 48-well crystal plate. The pooling solution consisted of 0.01 M Nickel chloride, 0.1 M Tris, and 20% PEG 2000 MME. Different concentrations of 1 μL protein, 0.2 μL of additive, and 1 μL of pooling solution were added to both sides of the pooling solution, resulting in a total of 48 additives being screened. The crystal plate was then sealed and crystals were grown at 18°C. Finally, BM3 protein crystals grew under conditions containing additives #8 and #36. Figure 6 As shown, the crystal form has changed, but the crystal quality is poor (8# is 0.1M Strontium chloride hexahydrate, 36# is 0.1M Taurine).

[0049] Comparative Example 4: BM3 protein was incubated with FAD, FMN, NADPH, and arachidonic acid before crystal screening.

[0050] The target protein obtained in step (3) of the same procedure as in Example 1 was incubated with FAD, FMN, NADPH and the substrate arachidonic acid for 2 hours, with a molar ratio of BM3:FAD:FMN:NADPH:arachidonic acid = 1:5:5:10:2. Afterwards, the concentrations of the incubated BM3 protein were adjusted to 5 mg / mL and 10 mg / mL, and then subjected to initial crystal screening by gas-phase diffusion. Specifically, 200 μL of pooling solution was added to each well of a 48-well crystal plate, and 1 μL of protein and 1 μL of pooling solution were added to both sides of the pooling solution. The crystal plate was then sealed and crystals were grown at 18°C. Finally, BM3 protein crystals grew under conditions containing 0.2 M Ammonium acetate, 0.15 M Magnesium acetate, 0.05 M HEPES Sodium salt, pH 7.0, and 5% (w / v) PEG 4000. Figure 7 As shown.

[0051] As can be seen from the changes in conditions in the examples and comparative examples that affect whether crystallization occurs and the quality of the crystals, the present invention provides a high-resolution method for easy full-chain crystallization. Unlike the general prior art theory that additives are the key to affecting crystal form and thus obtaining better crystal quality, the method of the present invention can obtain higher quality crystals without the influence of additives; and the operation of co-incubation with cofactors is decisive for the success of crystallization.

[0052] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for crystallizing BM3 protein from Bacillus megaterium, characterized in that, Includes the following steps: 1) The gene encoding the BM3 protein was constructed into a vector suitable for expression in E. coli and amplified in cells to obtain an expression plasmid; 2) The obtained expression plasmid was transformed into an E. coli host and cultured in liquid medium until the cell density OD reached 100%. 600 The concentration was 0.6-0.8, and then the culture temperature was lowered to 23℃ and expression was induced by isopropyl-β-D-thiogalactopyranoside IPTG at a final concentration of 0.4 mM. At the same time, 5-aminolevulinic acid 5-ALA and riboflavin were added to the induction culture system at a final concentration of 0.4 mM, and the culture was shaken at 160 rpm for 40 hours. 3) After cell harvesting, the recombinant BM3 protein with the fusion tag was purified by affinity chromatography, and the fusion tag was removed by a specific protease, followed by gel filtration and purification to a monodisperse state. 4) The purified BM3 protein, flavin cofactors FAD and FMN, and reduced nicotinamide adenine dinucleotide NADPH were incubated in a suitable buffer at 4°C for 2 h in the dark to obtain the cofactor-bound protein complex. 5) Crystallization was carried out using the sitting drop gas phase diffusion method. The crystallization temperature was 18℃, the pool liquid volume was 200 μL, and the drop volume was 1 μL protein solution plus 1 μL pool liquid. The pool liquid conditions for the growth of BM3 protein crystals after the initial screening were 0.01M nickel chloride, 0.1M Tris buffer and 20% PEG 2000 MME. The method can obtain BM3 protein crystals suitable for X-ray diffraction analysis.

2. The method for crystallizing BM3 protein from Bacillus megaterium according to claim 1, characterized in that, The expression vector contains an affinity tag to facilitate affinity chromatography purification and includes a protease cleavage site for removing the affinity tag.

3. The method for crystallizing BM3 protein from Bacillus megaterium according to claim 1, characterized in that, The affinity chromatography uses a binding buffer containing 20 mM Tris-HCl pH=7.5, 150 mM sodium chloride, and 5 mM imidazole, and an elution buffer containing 200 mM imidazole.

4. The method for crystallizing BM3 protein from Bacillus megaterium according to claim 1, characterized in that, The protease used for the protease digestion is TEV protease, and the digestion is carried out at 4-16℃, with a molar ratio of protease to target protein of 1:10-100.

5. The method for crystallizing BM3 protein from Bacillus megaterium according to claim 1, characterized in that, The purified protein was concentrated to 5-20 mg / ml for crystallization screening.

6. The method for crystallizing BM3 protein from Bacillus megaterium according to claim 1, characterized in that, The buffer solution was 20 mmol / L Tris, pH 7.5, and 150 mmol / L sodium chloride.

7. The method for crystallizing BM3 protein from Bacillus megaterium according to claim 1, characterized in that, Before the co-incubation step, a reducing agent of 1-5 mmol / L is added to the reaction system to keep the cofactor in a reduced or controlled oxidation state.

8. The method for crystallizing BM3 protein from Bacillus megaterium according to claim 1, characterized in that, The crystallization process employs micro-seedling to promote crystal growth.

9. A BM3 protein crystal, characterized in that, The crystal is prepared by the protein crystallization method according to any one of claims 1-8.

10. A BM3 protein crystal according to claim 9, characterized in that, The crystal exhibited diffraction capability in X-ray diffraction experiments, with its entire chain length suitable for three-dimensional structure analysis.

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