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CN-122012423-A - Method for crystallizing BM3 protein in bacillus megatherium

CN122012423ACN 122012423 ACN122012423 ACN 122012423ACN-122012423-A

Abstract

The invention discloses a method for crystallizing BM3 protein in bacillus megatherium, which comprises the steps of constructing a gene for encoding the BM3 protein into an expression vector, inducing expression in escherichia coli, purifying by affinity chromatography, cutting fusion tags, purifying by gel filtration, incubating with cofactors FAD, FMN and NADPH to form a compound, and crystallizing under the condition of specific pool liquid by a sitting-drop gas phase diffusion method.

Inventors

  • WANG JIA
  • SHI WENZHU
  • WANG XIAO
  • LIU LIN

Assignees

  • 安徽大学

Dates

Publication Date
20260512
Application Date
20260306

Claims (10)

  1. 1. The method for crystallizing BM3 protein in bacillus megatherium is characterized by comprising the following steps: 1) Constructing a gene encoding a BM3 protein into a vector suitable for expression in E.coli and amplifying in cells to obtain an expression plasmid; 2) Transferring the obtained expression plasmid into an escherichia coli host, culturing in a liquid culture medium until the cell density OD 600 is 0.6-0.8, then reducing the culture temperature to 23 ℃ and inducing the expression with isopropyl-beta-D-thiogalactoside IPTG with the final concentration of 0.4 mM, simultaneously adding 5-ALA of 5-aminolevulinic acid and Riboflavin with the final concentrations of 0.4 mM into an induction culture system, and culturing for 40 hours by shaking at 160 rpm; 3) Purifying recombinant BM3 protein with fusion tag by adopting affinity chromatography after harvesting cells, and purifying to a monodisperse state after cutting the fusion tag by using specific protease and gel filtration; 4) The molar ratio of purified BM3 protein to flavin cofactor FAD to FMN and reduced nicotinamide adenine dinucleotide NADPH is BM3 to FAD to FMN to NADPH=1 to 5 to 10 incubated in a suitable buffer in a 4 ℃ environment in the absence of light for 2h to obtain a cofactor-bound protein complex; 5) Crystallizing by adopting a sitting-drop gas phase diffusion method, wherein the crystallization temperature is 18 ℃, the volume of a pool solution is 200 mu L, 1 mu L of a protein solution is added into 1 mu L of the pool solution, and the conditions of the pool solution with BM3 protein crystal growth after preliminary screening are 0.01M, tris of nickel chloride buffer solution and 0.1M of 20% PEG 2000 MME; the method can obtain BM3 protein crystals suitable for X-ray diffraction analysis.
  2. 2. The method of claim 1, wherein the expression vector contains an affinity tag for affinity chromatography purification and a protease cleavage site for removal of the affinity tag.
  3. 3. The method of claim 1, wherein the affinity chromatography is performed with a binding buffer comprising 20 mM Tris-HCl ph=7.5, 150 mM sodium chloride and 5mM imidazole, and an elution buffer comprising 200 mM imidazole.
  4. 4. The method for crystallizing BM3 protein from bacillus megatherium as claimed in claim 1, wherein the protease used for the protease cleavage is TEV protease and the protease cleavage is carried out at 4-16 ℃ with a molar ratio of protease to target protein of 1:10-100.
  5. 5. The method for crystallizing BM3 protein from bacillus megatherium as claimed in claim 1, wherein said purified protein is concentrated to 5-20 mg/ml for crystallization screening.
  6. 6. The method for crystallizing BM3 protein from b.megaterium as claimed in claim 1, wherein the buffer is Tris 20 mmol/L, ph=7.5 and sodium chloride 150 mmol/L.
  7. 7. The method for crystallization of BM3 protein in b.megaterium according to claim 1, characterised in that 1-5mmol/L of reducing agent is added to the reaction system before the co-incubation step to keep the cofactor in reduced or controlled oxidation state.
  8. 8. The method for crystallization of BM3 protein in bacillus megatherium according to claim 1, wherein said crystallization is seeded with a trace amount to promote crystal growth.
  9. 9. A BM3 protein crystal prepared by the method of crystallization of a protein according to any one of claims 1 to 8.
  10. 10. The BM3 protein crystal according to claim 9, wherein said crystal exhibits a full chain length diffraction capability useful for three-dimensional structural resolution in X-ray diffraction experiments.

Description

Method for crystallizing BM3 protein in bacillus megatherium Technical Field The invention belongs to the technical field of structural biology, and particularly relates to a method for obtaining BM3 protein crystals in an expression system and BM3 protein crystals prepared by the method, which are suitable for protein three-dimensional structural analysis and functional mechanism research. Background The functional mechanism of the protein is hidden in the three-dimensional space structure, and the analysis of the structure of the protein is helpful to promote the understanding of the functional mechanism through the interaction between biomacromolecules, so as to solve some biological problems. The X-ray crystallography is the primary means of resolving protein structures. X-rays are high energy, short wavelength electromagnetic waves that produce diffraction effects by acting on electrons in the crystal when they strike molecular crystal particles. The diffraction signals are collected by the detector, so that the distribution of electron density in the crystal can be determined, and the position information of the particles is obtained, thereby providing basis for analyzing the crystal structure of the biomacromolecule. After crystallization of the protein, the three-dimensional structure can be seen by X-ray diffraction. However, this method cannot be used for analyzing proteins having a large molecular weight, and has a high requirement for protein crystallization, and therefore there is a limit to the structural analysis of proteins which cannot be crystallized. Cytochrome P450 is a super large family of proteases containing b-type heme, is widely found in organisms in nature, is one of the oldest and largest enzyme families, and is named cytochrome P450 because a complex formed by combining a reduced state of the protease with CO has a maximum absorption peak in the vicinity of 450 nm. BM3 is a classical self-sufficient monooxygenase in cytochrome P450, which is the 3 rd P450 enzyme found in Bacillus megaterium (Bacillus megaterium), designated BM3, which is a single-chain multi-domain enzyme with high stability, high catalytic activity and molecular weight of about 119: 119 kDa. BM3 protein, also called CYP102A1, is a kind of macromolecular motor-like fusion oxidase, and contains heme binding domain and flavin binding electron transfer domain, heme domain and reductase domain similar to mammal CPR, and has become research hot spot due to its unique structural and functional relationship in substrate oxidation and electron transfer. However, the complexity of the large molecular weight and structure thereof makes it difficult to obtain a high quality single crystal. The prior art reports structural information on the crystal structure of the different domains of BM3 and several truncated variants, but the full-length BM3 or the high-resolution crystal structure containing cofactor complexes in specific states is scarce. Conventional crystallization strategies include truncating the flexible fragment, introducing mutations to promote lattice contact, adding crystallization partners and cofactors to stabilize the conformation, and the like. There are also prior art techniques for resolving protein structures using cryoelectron microscopy, but this method relies on sample homogeneity and stability, and conformational heterogeneity of proteins in solution can reduce the final resolution. The art needs a high-quality protein crystallization method for analyzing the structure of BM3 protein in bacillus megatherium, and development of a set of expression purification and crystallization procedures which are stable, repeatable and suitable for obtaining diffractable crystals has important significance. Disclosure of Invention The invention aims to provide a method for preparing BM3 protein in an expression system and obtaining crystals for X-ray diffraction, which provides a structural basis for analyzing the full-length three-dimensional structure of BM3 and researching the electron transfer mechanism of BM3 protein, and provides BM3 protein crystals prepared by the method as a protection object. In order to achieve the above purpose, the present invention adopts the following technical scheme: The method for crystallizing BM3 protein in bacillus megatherium comprises the following steps: 1) Constructing a gene encoding a BM3 protein into a vector suitable for expression in E.coli and amplifying in cells to obtain an expression plasmid; 2) Transferring the obtained expression plasmid into an escherichia coli host, culturing in a liquid culture medium until the cell density OD 600 is 0.6-0.8, then reducing the culture temperature to 23 ℃ and inducing the expression with isopropyl-beta-D-thiogalactoside with the final concentration of 0.4 mM, and simultaneously adding 5-aminolevulinic acid and riboflavin with the final concentrations of 0.4 mM into an induction culture system respectively and culturing for 4