TAL1 and TAL1 S122A mutant recombinant protein as well as preparation method and application thereof

Abstract

The invention discloses TAL1 and TAL1S122A mutant recombinant proteins as well as a preparation method and application of the TAL1 and TAL1S122A mutant recombinant proteins. The amino acid sequence of the TAL1 and TAL1S122A mutant recombinant protein provided by the invention is as shown in SEQ ID NO: 1 or SEQ ID NO: 2. The recombinant protein can be used in in-vivo and in-vitro experiments of phosphorylation of TAL1 by MAP3K3 and other kinases. The invention also provides a preparation method of the recombinant protein. The preparation method comprises the steps of expression plasmid construction, expression test, amplification expression and affinity purification, protein dialysis, protein concentration and the like. The concentrated recombinant protein has the characteristics of high purity and good activity. In an in-vitro kinase experiment with a commercial MAP3K3 recombinant protein, the TAL1 recombinant protein can be smoothly phosphorylated by MAP3K3, and the phosphorylation degree of S122A mutation by MAP3K3 is reduced. The application has important significance for researching the TAL1 and the phosphorylation site of the TAL1.

Description

Technical Field

[0001] The present invention relates to a TAL1 and TAL1 S122A mutant recombinant protein and a preparation method and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] TAL1 (T-cell acute lymphoblastic leukemia protein 1) is a basic helix-loop-helix transcription factor that plays a role in a variety of biological processes, including regulation of embryonic hematopoiesis and control of cell proliferation and differentiation. TAL1 forms a heterodimer with E2A and HEB / HTF4 and binds to a DNA motif known as the E-box. Transgenic co-expression of TAL1 and the catalytic subunit of casein kinase IIalpha significantly accelerates leukemia development.

[0003] TAL1 can be phosphorylated on three highly conserved residues, Ser-122, Ser-172, or Thr-90. After phosphorylation, ubiquitin targets p-TAL1 and rapidly degrades it through the ubiquitin system.

[0004] Mitogen-activated protein kinase kinase 3 (MAP3K3) is a highly conserved member of the MAP3K superfamily with Ser / Thr protein kinase activity. MAP3K3 is involved in a variety of important biological pathways: activation of ERK5 and p38 in the MAPK pathway, regulation of the Toll-like receptor pathway and the NF-κB pathway, lymphocyte differentiation, and the development of cardiovascular and cerebrovascular diseases. MAP3K3 has been reported to be a key kinase in different cell types and inflammatory processes, such as neutrophils in acute lung injury (ALI) and platelets in myocardial infarction (MI). However, the role of MAP3K3 in phosphorylation of TAL1 is still unclear. Summary of the invention

[0005] The purpose of the present invention is to provide a TAL1 and TAL1 S122A mutant recombinant protein and a preparation method and application thereof. The TAL1 and TAL1 S122A mutant recombinant proteins prepared by the present invention have higher purity, and can be used in in vivo and in vitro experiments on TAL1 phosphorylation by MAP3K3 and other kinases, so as to better study the TAL1 phosphorylation mechanism and function.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a recombinant protein, which is a TAL1 recombinant protein or a TAL1S122A mutant recombinant protein, the amino acid sequence of the TAL1 recombinant protein is shown in SEQ ID NO: 1, and the amino acid sequence of the TAL1S122A mutant recombinant protein is shown in SEQ ID NO: 3.

[0008] Preferably, the recombinant protein is prepared by cloning the codon-optimized gene sequence of TAL1 recombinant protein or TAL1 S122A mutant recombinant protein into a pSmart-I (His-tag) expression vector with Kan resistance to construct an expression plasmid, the cloning site is BamHI-XhoI, and then undergoing Rosetta (DE3) strain expression test, strain culture and expression amplification, affinity purification, protein dialysis and protein concentration.

[0009] In a second aspect, the present invention provides a nucleotide for encoding the above-mentioned recombinant protein, the sequence of the nucleotide being as shown in SEQ ID NO: 2 or 4, wherein the nucleotide with the sequence as shown in SEQ ID NO: 2 is used to encode the TAL1 recombinant protein, and the nucleotide with the sequence as shown in SEQ ID NO: 4 is used to encode the TAL1S122A mutant recombinant protein.

[0010] In the third aspect, the present invention provides a method for preparing the above-mentioned recombinant protein, comprising: cloning the codon-optimized gene sequence of TAL1 recombinant protein or TAL1S122A mutant recombinant protein into a pSmart-I (His-tag) expression vector with Kan resistance to construct an expression plasmid, wherein the cloning site is BamHI-XhoI, and then preparing the protein through the steps of Rosetta (DE3) strain expression test, strain culture and expression amplification, affinity purification, protein dialysis and protein concentration.

[0011] In a fourth aspect, the present invention provides the use of the above recombinant protein in studying the mechanism and function of TAL1 and its phosphorylation site Ser-122 in vitro.

[0012] Preferably, the application includes: performing an in vitro kinase experiment on TAL1 recombinant protein, TAL1 S122A mutant recombinant protein and commercial MAP3K3 recombinant protein to study the effect of MAP3K3 recombinant protein on the phosphorylation of TAL1 Ser-122 site.

[0013] In a fifth aspect, the present invention provides use of a TAL1 S122A mutant recombinant protein in the preparation of a product for inhibiting phosphorylation of the TAL1Ser-122 site, wherein the amino acid sequence of the TAL1 S122A mutant recombinant protein is shown in SEQ ID NO:3.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The preparation method provided by the present invention prepares TAL1 recombinant protein and S122A mutant recombinant protein with higher purity and high activity, and provides an example of specific application in MAP3K3 kinase experiments, discovering the phosphorylation effect of MAP3K3 on TAL1 at the S122 site; this application is of great significance for further research on TAL1 and its phosphorylation sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : TAL1 recombinant protein expression test SDS-PAGE results; where, M: Marker; Lane 1: before 37℃ induction; Lane 2: after 37℃ induction; Lane 3: before 16℃ induction; Lane 4: after 16℃ induction;

[0017] Figure 2 : TAL1 recombinant protein affinity purification SDS-PAGE results; where M: Marker; Lane 1: supernatant protein; Lane 2: flow-through; Lane 3-5: elution samples;

[0018] Figure 3 : TAL1 recombinant protein concentration results; M: Marker; Lane 1: concentrated sample;

[0019] Figure 4 : TAL1 S122A mutant recombinant protein expression test SDS-PAGE results; where, M: Marker; Lane 1: before 37℃ induction; Lane 2: after 37℃ induction; Lane 3: before 16℃ induction; Lane 4: after 16℃ induction;

[0020] Figure 5 :The results of affinity purification of TAL1 S122A mutant recombinant protein by SDS-PAGE; where M: Marker; Lane 1: supernatant protein; Lane 2: flow-through; Lane 3-5: elution samples;

[0021] Figure 6 :Concentration results of TAL1 S122A mutant recombinant protein; where M: Marker; Lane 1: concentrated sample;

[0022] Figure 7 : Kinase assay of MAP3K3 recombinant protein, TAL1 recombinant protein and S122A mutant recombinant protein. DETAILED DESCRIPTION

[0023] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0024] The present invention provides a TAL1 recombinant protein and its S122A mutant recombinant protein and a preparation method thereof, wherein the preparation method comprises the steps of expression plasmid construction, expression test, amplified expression and affinity purification, protein dialysis, protein concentration and the like. After concentration, the TAL1 recombinant protein and the S122A mutant recombinant protein exhibit high purity and good activity. In an in vitro kinase experiment with commercialized MAP3K3 recombinant protein, the TAL1 recombinant protein can be successfully phosphorylated by MAP3K3, while the S122A mutation is phosphorylated less by MAP3K3.

[0025] Example 1: Preparation and purification of TAL1 recombinant protein

[0026] 1) Expression plasmid construction: This example uses the Kan-resistant pSmart-I (His-tag) expression vector, the cloning site is BamHI-XhoI, and the expression strain is Rosetta (DE3). The TAL1 recombinant protein sequence is shown in SEQ ID ID: 1:

[0027] MTERPPSEAARSDPQLEGRDAAEASMAPPHLVLLNGVAKETSRAAAAEPPVIE

[0028] LGARGGPGGGPAGGGGAARDLKGRDAATAEARHRVPTTELCRPPGPAPAPAP

[0029] ASVTAELPGDGRMVQLSPPALAAPAAPGRALLYSLSQPLASLGSGFFGEPDAF

[0030] PMFTTNNRVKRRPSPYEMEITDGPHTKVVRRIFTNSRERWRQQNVNGAFAEL

[0031] RKLIPTHPPDKKLSKNEILRLAMKYINFLAKLLNDQEEEGTQRAKTGKDPVVG

[0032] AGGGGGGGGGGGAPPDDLLQDVLSPNSSCGSSLDGAASPDSYTEEPAPKHTARSLHPAMLPAADGAGPR. The target gene was codon optimized according to the preferred codons of the expression host. The gene sequence after codon optimization is shown in SEQ ID ID: 2:

[0033] ATGACCGAACGTCCACCGTCTGAAGCTGCACGTTCTGATCCGCAGCTGGAA

[0034] GGTCGTGATGCAGCTGAAGCGTCTATGGCACCACCGCATCTGGTTCTGCTG

[0035] AACGGTGTTGCGAAAGAAACCTCTCGTGCTGCTGCTGCTGAACCGCCGGT

[0036] TATCGAACTGGGTGCTCGTGGTGGTCCAGGTGGTGGTCCAGCAGGTGGTG

[0037] GTGGTGCTGCACGTGACCTGAAAGGTCGTGATGCTGCTACTGCTGAAGCTC

[0038] GTCATCGTGTTCCGACCACCGAACTGTGTCGTCCACCGGGTCCAGCACCAG

[0039] CACCAGCTCCAGCGTCTGTTACCGCTGAACTGCCGGGTGATGGTCGTATGG

[0040] TTCAGCTGAGTCCGCCAGCTCTGGCAGCACCAGCTGCTCCAGGTCGTGCTC

[0041] TGCTGTACTCTCTGTCTCAGCCGCTGGCTAGTCTGGGTTCTGGTTTCTTCGG

[0042] TGAACCGGATGCGTTTCCGATGTTCACCACCAACAACCGTGTTAAACGTCG

[0043] TCCATCTCCGTACGAAATGGAAATCACCGATGGTCCGCACACCAAAGTTGT

[0044] TCGTCGTATCTTCACCAACTCTCGTGAACGTTGGCGTCAGCAGAACGTTAA

[0045] CGGTGCTTTCGCAGAACTGCGTAAACTGATTCCGACTCATCCGCCGGACAA

[0046] GAAGCTGTCTAAGAACGAAATTCTGCGTCTGGCGATGAAATACATCAACTT

[0047] TCTGGCGAAACTGCTGAACGACCAAGAAGAAGAAGGTACTCAGCGTGCTA

[0048] AGACCGGCAAAGATCCGGTTGTTGGTGCTGGTGGTGGTGGTGGTGGTGGT

[0049] GGTGGTGGTGCACCACCGGATGATCTGCTGCAAGACGTTCTGTCTCCGAAC

[0050] TCTAGCTGCGGTTCTAGCCTGGATGGTGCAGCTTCTCCGGACTCTTACACC

[0051] GAAGAACCGGCTCCGAAACACACCGCTCGTTCTCTGCATCCGGCTATGCTGCCAGCTGCTGATGGTGCTGGTCCGCGT. After codon optimization, restriction enzyme sites were introduced at both ends of the target gene, primers were designed for PCR amplification, reaction products were detected by gel electrophoresis and PCR products were recovered by gel; the empty plasmid vector was treated with double enzyme digestion, and the treated vector was recovered by gel; the gel recovery product and the linearized vector were subjected to recombination ligation reaction; the recombinant ligation product was transformed into competent cells, and 4 single colonies were randomly selected for positive screening. The colonies identified as positive by PCR were cultured at 37°C overnight, glycerol bacteria were preserved, and plasmids were extracted for sequencing identification.

[0052] 2) Expression test: Transform the expression strain: Take out the competent cells from the ultra-low temperature refrigerator and place them on crushed ice for 5 minutes; add 100ng of plasmid DNA to the competent cells and mix gently. After incubating on ice for 30 minutes, heat shock in a 42℃ water bath for 90 seconds and place on ice for 5 minutes. Add 900μl of room temperature anti-free LB medium, culture at 37℃ and 220rpm for 45-60 minutes, centrifuge at 4500rpm for 2 minutes, carefully remove the supernatant, gently mix the remaining liquid with a pipette, pipette it onto the LB solid plate, spread it evenly, invert the plate and culture it overnight at 37℃; inoculate the strain into LB medium, culture at 37℃ and 220rpm until the bacterial OD600 is 0.4-0.6, add IPTG to the culture to a final concentration of 1.0mM, continue to culture at 37℃ and 220rpm for 4h, induce the expression of the fusion protein, collect the bacteria and prepare samples for SDS-PAGE analysis; Inoculate the strain into LB medium in another tube, and culture at 37℃220rpm until the bacterial OD600 is 0.4-0.6. Add IPTG to the culture to a final concentration of 0.2mM, and continue to culture at 16℃220rpm for 16h to induce the expression of fusion protein. Collect the bacteria and prepare samples for SDS-PAGE detection. 1.1.3 Pipette 900μl of the induced culture, collect the bacteria by centrifugation, resuspend the precipitate with 900μl1xPBS, and perform ultrasonic lysis. Centrifuge at 12000rpm for 5min to distinguish the supernatant protein and inclusion body protein, and prepare samples for SDS-PAGE detection. The results are as follows: Figure 1 shown.

[0053] 3) Amplification and affinity purification: Inoculate the strain into 400 ml LB medium, culture at 37°C 220 rpm until the bacterial OD600 is 0.4-0.6, add IPTG to the culture to a final concentration of 1.0 mM, continue to culture at 37°C 220 rpm for 4 hours, and collect the bacteria by high-speed centrifugation; resuspend the bacteria in 30 ml 1xPBS, add 30 μl β-mercaptoethanol and 300 μl PMSF, ultrasonically break in an ice bath, ultrasonically pause for 3 seconds, and ultrasonicate 100 times. Centrifuge at 12000 rpm, 4°C for 15 minutes, and aspirate the supernatant protein into a new tube. Ni-Smart affinity chromatography to purify supernatant protein: (equilibrium buffer: 20mM PB, 0.5M NaCl, pH 7.4; washing buffer: 20mM PB, 0.5M NaCl, 10mM Imidazole, pH 7.4; elution buffer: 20mM PB, 0.5M NaCl, 100mM Imidazole, pH 7.4), (3.0ml filler, loading capacity ~8mg / ml with His-tag protein, flow rate of about 1ml / min, room temperature column) 15ml deionized water to wash the column, wash once; 15ml equilibrium buffer, balance once; load, repeat loading once, collect the penetration and retain 20μl of the sample for electrophoresis detection; 15ml washing buffer to wash the column, collect the washing solution and retain 20μl of the sample for electrophoresis detection; 15ml elution buffer to elute the protein, collect the eluate and retain 20μl of the sample for electrophoresis detection; 15ml deionized water to wash the column, wash once; 15ml0.5M Wash the column with NaOH; wash the column with 15 ml deionized water for three times; balance the column with 20% ethanol and store it. Prepare the purified sample for SDS-PAGE detection, select the qualified eluent for dialysis and concentration, and the results are as follows: Figure 2 shown.

[0054] 4) Protein dialysis: The qualified eluted sample was dialyzed into 1xPBS, 10% Glycerol, pH 7.4 solution at 4°C overnight. The target protein was clarified without precipitation.

[0055] 5) Protein concentration: Centrifuge at 4500 rpm, 4°C, use ultrafiltration tube to concentrate the target protein to a suitable concentration, take a sample for SDS-PAGE, and determine the final protein concentration and protein amount. The results are as follows: Figure 3 shown.

[0056] The results showed that the TAL1 recombinant protein was well expressed, had high purity after purification, and had high activity after concentration.

[0057] Example 2: Preparation and purification of TAL1 S122A mutant recombinant protein

[0058]

[0059] The results showed that the TAL1 recombinant protein was well expressed, had high purity after purification, and had high activity after concentration. Figure 4 Shown are the SDS-PAGE results of the TAL1 S122A mutant recombinant protein expression test; Figure 5 Shown are the SDS-PAGE results of affinity purification of TAL1 S122A mutant recombinant protein; Figure 6 Shown are the results of TAL1 S122A mutant recombinant protein enrichment.

[0060] Example 3: Kinase assay of MAP3K3 recombinant protein, TAL1 recombinant protein and S122A mutant recombinant protein

[0061] This example uses recombinant human MAP3K3 protein and recombinant SUMO-His-tagged TAL1 (WT or mutated to S122A) in 1X kinase buffer (diluted from 10x kinase buffer) at 30°C with 250μM ATP for 30 minutes. The reaction was stopped by adding 1xSDS loading buffer. The samples were boiled for 5 minutes. The proteins were separated by SDS-PAGE and analyzed by western blotting.

[0062] The results confirmed that MAP3K3 can phosphorylate TAL1, and the phosphorylation level is weakened after S122A mutation, such as Figure 7 shown.

[0063] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A recombinant protein, characterized in that The recombinant protein is a TAL1 recombinant protein or a TAL1S122A mutant recombinant protein. The amino acid sequence of the TAL1 recombinant protein is shown in SEQ ID NO: 1, and the amino acid sequence of the TAL1 S122A mutant recombinant protein is shown in SEQ ID NO:

3.

2. The recombinant protein according to claim 1, characterized in that The recombinant protein is prepared by cloning the codon-optimized gene sequence of TAL1 recombinant protein or TAL1S122A mutant recombinant protein into a pSmart-I (His-tag) expression vector with Kan resistance to construct an expression plasmid, with the cloning site being BamHI-XhoI, and then undergoing Rosetta (DE3) strain expression testing, strain culture and expression amplification, affinity purification, protein dialysis and protein concentration.

3. The nucleotide encoding the recombinant protein according to claim 1, characterized in that: The sequence of the nucleotide is as shown in SEQ ID NO: 2 or 4, wherein the nucleotide sequence as shown in SEQ ID NO: 2 is used to encode the TAL1 recombinant protein, and the nucleotide sequence as shown in SEQ ID NO: 4 is used to encode the TAL1 S122A mutant recombinant protein.

4. The method for preparing the recombinant protein according to claim 1, characterized in that: include: The codon-optimized gene sequence of TAL1 recombinant protein or TAL1S122A mutant recombinant protein was cloned into the pSmart-I (His-tag) expression vector with Kan resistance to construct an expression plasmid with the cloning site of BamHI-XhoI. The expression plasmid was then prepared through the steps of Rosetta (DE3) strain expression test, strain culture amplification expression, affinity purification, protein dialysis and protein concentration.

5. Use of the recombinant protein of claim 1 in studying the mechanism and function of TAL1 and its phosphorylation site Ser-122 in vitro.

6. The use according to claim 5, characterized in that The application includes: performing an in vitro kinase experiment on TAL1 recombinant protein, TAL1 S122A mutant recombinant protein and commercial MAP3K3 recombinant protein to study the effect of MAP3K3 recombinant protein on the phosphorylation of TAL1 Ser-122 site.

7. Use of TAL1 S122A mutant recombinant protein in the preparation of a product for inhibiting TAL1 Ser-122 site phosphorylation, characterized in that: The amino acid sequence of the TAL1 S122A mutant recombinant protein is shown in SEQ ID NO:3.

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