CN114920815A - Method for promoting crystallization of transcription factor GATA6 protein - Google Patents
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Abstract
The invention relates to a method for promoting crystallization of transcription factor GATA6 protein, belongs to the technical field of structure biology, solves the technical problem that the structure is not resolved due to low expression, easy sedimentation and no crystallization of GATA6 protein, and comprises the following steps: the clone construction of GATA6-DBD → expression of GATA6-DBD protein → purification of GATA6-DBD protein → crystallization of GATA6-DBD protein and DNA complex, and the complex crystal of GATA6-DBD and DNA. According to the invention, the three-dimensional space structure of interaction between GATA6 and a promoter of a cardiomegative embryonic gene is analyzed through a GATA6 protein crystallization promotion experiment, so that detailed information of a compound combination mode is provided, the structural problem of cardiomegative promotion is solved, and an accurate target is provided for screening possible drugs for inhibiting cardiomegative hypertrophy.
Description
Technical Field
The invention belongs to the technical field of structural biology, and particularly relates to a method for promoting crystallization of a transcription factor GATA6 protein.
Background
The three-dimensional structure of the protein can provide detailed information of the interaction between the biological macromolecules and can also observe the dynamic change of the biological macromolecules when the biological macromolecules perform the functions of the biological macromolecules. The analysis of the structure of the protein can not only promote the understanding of biological functions and molecular mechanisms and explain important biological problems, but also explore the pathogenesis of diseases related to the biological macromolecular dysfunction and design the targeted drugs of the related diseases. The GATA6 is a transcription factor belonging to a member of the GATA protein family, and is a transcription factor of GATA6 activated in myocardial hypertrophy diseases, because its DNA-binding domain (DBD) is directly bound to a promoter of a gene of a cardiac hypertrophy-causing embryo to cause myocardial hypertrophy. Therefore, it has important theoretical and clinical significance to explore the pathogenesis of myocardial hypertrophy by taking the GATA6 transcription factor as a structural basis and develop a new drug for inhibiting myocardial hypertrophy.
The structure of GATA6 has not been resolved so far, and the structural biological mechanism of its binding to promoter promoting myocardial hypertrophy is not clear:
(1) the GATA6 structure is required for drug development, but other analyzed structures of the GATA family, such as GATA1 and GATA3, can be used for reference, but the GATA6 mainly plays a role in the hematopoietic system, and the GATA6 mainly participates in cardiac development, so that the analysis of the structure of the GATA6 is very necessary;
(2) GATA6 is particularly easy to precipitate in the protein purification process, so that single and high-purity protein is difficult to obtain, and the subsequent protein crystallization experiment is difficult to analyze the spatial structure;
(3) GATA6, which is difficult to obtain protein crystals during crystallization, was not able to be subjected to further X-ray diffraction experiments.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, solve the technical problem that the structure is not resolved due to low GATA6 protein expression, easy sedimentation and no crystallization, and provide a method for promoting the crystallization of a transcription factor GATA6 protein, wherein a GATA6 protein crystallization promotion experiment is used for analyzing the three-dimensional space structure of the interaction between GATA6 and a promoter of a cardiac hypertrophy causing embryonic gene so as to provide detailed information of a compound binding mode, understand the structural problem of cardiac hypertrophy promotion, and provide a precise target for screening possible drugs for inhibiting cardiac hypertrophy.
The design concept of the invention is as follows: inspired by structural biology and molecular biology, the DBD sequence of GATA6 is cloned into a vector suitable for GATA6 expression, and the vector needs to meet the requirements of large-scale expression of GATA6 and reduction of GATA6 sedimentation. In addition, after the protein is successfully expressed, the GATA6-DBD and DNA are required to be incubated, a compound crystal combining the GATA6-DBD and the DNA is obtained through a eutectic experiment, the interaction mode of the GATA6-DBD and the DNA is determined after analysis, a basis is provided for possible pathological processes, and the method has important significance for screening clinical drugs of related diseases by taking the GATA6 as a target protein.
The invention is realized by the following technical scheme:
the invention provides five DNA sequences capable of being combined with a GATA6-DBD region, and further obtains a compound crystal of the DNA and the GATA6-DBD in a compound crystallization experiment of the DNA and the GATA6-DBD, thereby providing a basis for subsequent protein structure analysis. The method comprises the following steps:
cloning construction of S1 and GATA 6-DBD: performing PCR amplification on a human GATA6 (hGATA 6[ NM-005257.6 ]) gene to obtain a GATA6-DBD target fragment, connecting the GATA6-DBD target fragment to a pET32M.3C vector, transforming the GATA target fragment to DH5 alpha competent cells, plating and inoculating bacteria, culturing the cells overnight at the temperature of 37 ℃ and 220rpm, and then extracting recombinant plasmids for double enzyme digestion identification and sequencing;
s2, GATA6-DBD protein expression: transferring the recombinant plasmid with correct sequencing in the step S1 into escherichia coliBL21(DE3)Plating and inoculating bacteria in competent cells, culturing overnight at 37 ℃ and 220rpm, adding IPTG (isopropyl-beta-D-thiogalactoside) to induce for 16h at 16 ℃, and collecting bacteria for later use;
s3, GATA6-DBD protein purification: and (4) re-suspending the bacterial cells obtained in the step S2 in a phosphate buffer, adding a Dnase solution with a concentration of 10 mug/mL, an Rnase solution with a concentration of 10 mug/mL, glycerol with a concentration of 5% and a PMSF solution with a concentration of 0.5mM into the bacterial cells before ultrasonic disruption, wherein the volume ratio of the Dnase solution to the Rnase solution to the glycerol to the PMSF solution to the bacterial cells is 1: 1: 50: 5: 1000, crushing thalli by using an ultrasonicator, centrifuging at the overspeed of 15000rpm under the temperature environment of 4 ℃ to obtain supernatant, incubating the supernatant with a Ni medium, then performing affinity chromatography purification, sequentially performing ion exchange chromatography and gel filtration chromatography on target protein obtained by elution, finally detecting by using SDS-PAGE electrophoresis, and collecting GATA6-DBD target protein;
s4, GATA6-DBD protein and DNA complex crystallization: and (3) co-incubating the GATA6-DBD target protein collected in the step S3 and chemically synthesized DNA containing a GATA site promoter partial sequence, and performing eutectic culture to prepare a GATA6-DBD and DNA combined compound crystal.
Further, in the step S4, the GATA6-DBD and the DNA complex crystal are obtained by gas phase diffusion screening, which comprises the following steps:
(1) preparation of dsDNA:
two chemically synthesized single-stranded promoters containing GATA sites are used to contain ZnCl 2 、MgCl 2 Diluting a mixed solution of DTT, Tris-HCl, NaCl and 5% glycerol, annealing at 100 ℃ for 5min, standing to room temperature, and mixing with GATA6 according to a molar ratio (0.5-1): 1 incubation on ice for at least 30 min;
(2) and crystal screening and optimizing the compound:
firstly, screening crystals of the compound by adopting a gas phase diffusion method:
the initial crystallization conditions were screened using a crystallography kit of CrystalScareningKitI & II, IndexKit or PGERXKit, manufactured by HamptonResearch company, comprising the steps of: adding 1 mu L of protein into the center of a cover glass, adding 1 mu L of pool liquid, mixing and inverting the mixture on a hanging drop crystal plate, adding 200 mu L of pool liquid into each hole, sealing the hole with vacuum lipid, standing the hole in a constant temperature crystal incubator at 16 ℃ for crystal culture, and obtaining a GATA6-DBD compound primary screening crystal;
and secondly, optimizing the primary screened crystal:
optimizing the primary screened crystal by a protein concentration gradient, primary screening condition pH, salt concentration, precipitant concentration, adding additive or iterative refining optimization method to obtain the GATA6-DBD compound optimized crystal.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a reliable and efficient protein crystal for successfully analyzing the interaction three-dimensional space structure of GATA6 and DNA. In the invention, a GATA6-DBD sequence is selected by molecular cloning, is constructed into a vector with the most suitable expression, and is then transformed into Escherichia coli, so that the problem of protein precipitation is solved. In addition, the problem of the GATA6-DBD protein not crystallizing is solved by incubation with chemically synthesized dsDNA containing GATA binding sites. The crystal is further optimized through subsequent steps, and the crystal meeting the diffraction condition is obtained.
In a word, the invention provides a structural basis for analyzing the structure of the GATA6-DBD complex, understanding the pathological process of myocardial hypertrophy and screening effective medicaments.
Drawings
FIG. 1 is a flow chart of protein purification according to the present invention;
FIG. 2 is an agarose gel electrophoresis gel of a plasmid successfully cloned with GATA6-DBD molecule for double restriction enzyme validation;
FIG. 3 shows the GATA6-DBD protein in E.coliBL21（DE3）Protein coomassie brilliant blue gel images before and after test expression;
FIG. 4 is a chromatogram and a protein gel of the GATA6-DBD protein after three-step purification;
FIG. 5 is a chromatogram of gel filtration after incubation of GATA6-DBD with dsDNA;
FIG. 6 is a graphic representation of the prescreened crystals of the GATA6-DBD complex;
FIG. 7 is a diagram of the optimized crystal of the GATA6-DBD complex.
Detailed Description
The invention is described in further detail below with reference to the figures and examples.
As shown in FIG. 1, in the method for promoting the crystallization of the transcription factor GATA6 protein, five DNA sequences capable of binding with the GATA6-DBD region are used in the embodiment, and further in the experiment of crystallizing the complex of the DNA and the GATA6-DBD, a complex crystal of the two is obtained, so that a basis is provided for the subsequent protein structure analysis. The method comprises the following steps:
cloning construction of S1 and GATA 6-DBD: performing PCR amplification on a human GATA6 (hGATA 6[ NM _005257.6 ]) gene to obtain a GATA6-DBD target fragment, connecting the fragment to a pET32M.3C vector, transforming the fragment to a DH5 alpha competent cell, plating and inoculating the cell, culturing the cell overnight at 220rpm at 37 ℃, extracting a recombinant plasmid, and performing double-enzyme digestion identification and sequencing, wherein the double-enzyme digestion identification result is shown in figure 2;
s2, GATA6-DBD protein expression: transferring the recombinant plasmid with correct sequencing in the step S1 into escherichia coliBL21(DE3)Plating and inoculating bacteria in competent cells, culturing at 37 ℃ and 220rpm overnight, adding IPTG (isopropyl-beta-thiogalactoside) to induce at 16 ℃ for 16h, confirming that protein is different before and after IPTG induction from figure 3, and collecting thalli for later use;
s3, GATA6-DBD protein purification: and (4) resuspending the thallus obtained in the step (S2) in a phosphate buffer solution, adding a Dnase solution with the concentration of 10 mug/mL, an Rnase solution with the concentration of 10 mug/mL, glycerol with the concentration of 5% and a PMSF solution with the concentration of 0.5mM into the bacteria solution before ultrasonic crushing, wherein the volume ratio of the Dnase solution to the Rnase solution to the glycerol to the PMSF solution to the bacteria solution is 1: 1: 50: 5: 1000, crushing thalli by using an ultrasonicator, centrifuging at the overspeed of 15000rpm under the temperature environment of 4 ℃ to obtain supernatant, incubating the supernatant with a Ni medium, then performing affinity chromatography purification, sequentially performing ion exchange chromatography and gel filtration chromatography on target protein obtained by elution, finally performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoresis detection, and collecting GATA6-DBD target protein supernatant, wherein the target protein has high purity and good uniformity;
s4, GATA6-DBD protein and DNA complex crystallization: and (3) incubating the GATA6-DBD protein collected in the step S3 with chemically synthesized DNA containing the GATA site promoter partial sequence, verifying the successful combination of the GATA6-DBD protein and the chemically synthesized DNA by gel filtration chromatography after incubation as shown in figure 5, and performing eutectic culture to prepare the GATA6-DBD and DNA combined compound crystal.
Further, in the step S4, the GATA6-DBD and the DNA complex crystal are obtained by gas phase diffusion screening, which comprises the following steps:
(1) preparation of dsDNA:
chemically synthesized organic compounds containingPromoter of GATA site with ZnCl-containing single strand 2 、MgCl 2 Diluting a mixed solution of DTT, Tris-HCl, NaCl and 5% glycerol, annealing at 100 ℃ for 5min, standing to room temperature, and mixing with GATA6 according to a molar ratio (0.5-1): 1 incubation on ice for at least 30min, involving five different DNAs in this step;
(2) and crystal screening and optimizing the compound:
firstly, screening crystals of the compound by adopting a gas phase diffusion method:
the initial crystallization conditions were screened using a crystallography kit of CrystalScreeningKitI & II, IndexKit or PGERXKit, manufactured by HamptonResearch, Inc., comprising the following steps: adding 1 mu L of protein into the center of a cover glass, adding 1 mu L of pool liquid, mixing and inverting the mixture on a hanging drop crystal plate, adding 200 mu L of pool liquid into each hole, sealing the hole with vacuum lipid, standing the hole in a constant temperature crystal incubator at 16 ℃ for crystal culture, and obtaining a GATA6-DBD compound prescreened crystal as shown in FIG. 6;
secondly, optimizing the primary screened crystal:
optimizing the primary-screened crystal by protein concentration gradient, primary screening condition pH, salt concentration, precipitant concentration, adding additive or iterative refining optimization method to obtain the GATA6-DBD compound optimized crystal, as shown in FIG. 7.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (2)
1. A method for promoting crystallization of transcription factor GATA6 protein, comprising the steps of:
cloning construction of S1 and GATA 6-DBD: performing PCR amplification on a human GATA6 (hGATA 6[ NM-005257.6 ]) gene to obtain a GATA6-DBD target fragment, connecting the GATA6-DBD target fragment to a pET32M.3C vector, transforming the GATA target fragment to a DH5 alpha competent cell, plating and inoculating bacteria, culturing the cells at 37 ℃ and 220rpm overnight, and then extracting a recombinant plasmid for double enzyme digestion identification and sequencing;
s2, GATA6-DBD protein expression: transferring the recombinant plasmid with correct sequencing in the step S1 into escherichia coliBL21(DE3)Plating and inoculating bacteria in competent cells, culturing overnight at 37 ℃ and 220rpm, adding IPTG (isopropyl-beta-D-thiogalactoside) and inducing for 16h at 16 ℃, and then collecting bacteria for later use;
s3, GATA6-DBD protein purification: and (4) resuspending the thallus obtained in the step (S2) in a phosphate buffer solution, adding a Dnase solution with the concentration of 10 mug/mL, an Rnase solution with the concentration of 10 mug/mL, glycerol with the concentration of 5% and a PMSF solution with the concentration of 0.5mM into the bacteria solution before ultrasonic crushing, wherein the volume ratio of the Dnase solution to the Rnase solution to the glycerol to the PMSF solution to the bacteria solution is 1: 1: 50: 5: 1000, crushing thalli by using an ultrasonicator, centrifuging at the overspeed of 15000rpm under the temperature environment of 4 ℃ to obtain supernatant, incubating the supernatant with a Ni medium, then performing affinity chromatography purification, sequentially performing ion exchange chromatography and gel filtration chromatography on target protein obtained by elution, finally detecting by using SDS-PAGE electrophoresis, and collecting GATA6-DBD target protein;
s4, GATA6-DBD protein and DNA complex crystallization: and (3) co-incubating the GATA6-DBD protein collected in the step (S3) with chemically synthesized DNA containing a GATA site promoter partial sequence, and performing eutectic culture to obtain a GATA6-DBD and DNA-bound compound crystal.
2. The method for promoting crystallization of transcription factor GATA6 protein according to claim 1, wherein: in the step S4, the GATA6-DBD and the DNA compound crystal are obtained by screening through a gas phase diffusion method, which comprises the following steps:
(1) preparation of dsDNA:
two chemically synthesized single-stranded DNAs of a GATA site-containing promoter are treated with ZnCl 2 、MgCl 2 Diluting a mixed solution of DTT, Tris-HCl, NaCl and 5% glycerol, annealing at 100 ℃ for 5min, standing to room temperature, and mixing with GATA6 according to a molar ratio (0.5-1): 1 incubation on ice for at least 30 min;
(2) and crystal screening and optimizing the compound:
firstly, screening crystals of the compound by adopting a gas phase diffusion method:
the initial crystallization conditions were screened using a crystallography kit of CrystalScreeningKitI & II, IndexKit or PGERXKit, manufactured by HamptonResearch, Inc., comprising the following steps: adding 1 mu L of protein in the center of a cover glass, adding 1 mu L of pool liquid, mixing and inverting the mixture on a hanging drop crystal plate, adding 200 mu L of pool liquid into each hole, sealing the holes with vacuum lipid, standing the holes in a constant temperature crystal incubator at 16 ℃ for crystal cultivation, and obtaining a GATA6-DBD compound primary screening crystal;
secondly, optimizing the primary screened crystal:
optimizing the primary screened crystal by protein concentration gradient, primary screening condition pH, salt concentration, precipitant concentration, addive or iterative searching optimization method to obtain the GATA6-DBD compound optimized crystal.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111675754A (en) * | 2020-05-19 | 2020-09-18 | 四川一埃科技有限公司 | Botulinum toxin protein crystal structure, crystal preparation method and resolution method |
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| US20210254012A1 (en) * | 2018-06-09 | 2021-08-19 | Arizona Board Of Regents On Behalf Of Arizona State University | Next generation designer liver organoids and their methods of preparation and use |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20210254012A1 (en) * | 2018-06-09 | 2021-08-19 | Arizona Board Of Regents On Behalf Of Arizona State University | Next generation designer liver organoids and their methods of preparation and use |
| CN111675754A (en) * | 2020-05-19 | 2020-09-18 | 四川一埃科技有限公司 | Botulinum toxin protein crystal structure, crystal preparation method and resolution method |
| CN112159800A (en) * | 2020-08-10 | 2021-01-01 | 深圳晶蛋生物医药科技有限公司 | PARP15 protein and Niraparib inhibitor compound crystal and preparation method thereof |
Non-Patent Citations (3)
| Title |
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| CHAO-CHUNG KUO,ET AL: "Detection of RNA–DNA binding sites in long noncoding RNAs", NUCLEIC ACIDS RESEARCH * |
| VON DER FAKULTÄT FÜR MATHEMATIK，ET AL: "Computational Detection of Triple Helix Binding Domains in Long Non-Coding RNAs", HTTP://PUBLICATIONS.RWTH-AACHEN.DE/RECORD/766795/FILES/766795.PDF * |
| 谭鹏;徐曼;梁航华;张亚军;胡仁建;崔红娟;: "家蚕GATA转录因子BmGATA6的克隆、多克隆抗体制备及组织细胞定位", 中国农业科学 * |
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