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CL7 protein, high-activity recombinant TET enzyme CL7-NgTET1, prokaryotic expression vector and application thereof

A prokaryotic expression, cl7-ngtet1 technology, applied in the biological field, can solve the problems of limited application prospects, low accuracy, and low efficiency of NgTET1, and achieve the effects of improving biological activity in vitro, improving oxidation capacity, and solving low yield

Pending Publication Date: 2022-03-01
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For application markets such as tumor early screening that strictly require methylation detection, low conversion means low efficiency and low accuracy, and the original active NgTET1 has limited application prospects

Method used

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  • CL7 protein, high-activity recombinant TET enzyme CL7-NgTET1, prokaryotic expression vector and application thereof
  • CL7 protein, high-activity recombinant TET enzyme CL7-NgTET1, prokaryotic expression vector and application thereof
  • CL7 protein, high-activity recombinant TET enzyme CL7-NgTET1, prokaryotic expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Construction of recombinant CL7-NgTET1 vector

[0026] (1) Gene synthesis: synthesize according to the protein sequences of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 as templates;

[0027] (2) Primer design: According to the two ends of the above fragments and the two ends of the pET28a vector entry point, about 20 bp respectively, the amplification and assembly primers were designed as homology arms. The primer sequences are as follows:

[0028] M-Forward: 5'-GTGCCGCGCGGCAGCCATATGAAAATCGAAGAAGGT-3',

[0029] M-Reverse: 5'-CGGTTCGTTAGATTTGGAACCCTGAAAATACAGATT-3',

[0030] CN-Forward: 5'-AATCTGTATTTTCAGGGTTCCAAAATCTAACGAACCG-3'

[0031] CN-Reverse: 5'-TGCTCGAGTGCGGCCGCTTATTTGGTTTCTTTATGA-3'

[0032] V-Forward: 5'-ATAAGCGGCCGCACTCGAGCA-3'

[0033] V-Reverse: 5'-ACCTTCTTCGATTTTCATATGG-3'

[0034] (3) Gene amplification and gel recovery: Use the KOD high-fidelity DNA polymerase amplification system, add the above primers and templates to form a 50uL reactio...

Embodiment 2

[0038] Example 2: SDS-PAGE and enzyme digestion verification of the expression of recombinant highly active CL7-NgTET1 in Rossetta (DE3)

[0039] The specific implementation steps are as follows:

[0040] (1) Conversion: From Take out Rossetta (DE3) competent cells in the ultra-low temperature refrigerator Thaw on ice and add ligation product keep on ice Heat shock at 42°C in a water bath Place on ice again join in No anti-LB liquid medium at room temperature, placed in a shaker at 37°C for 60 min at a speed of 180r / min; mix the obtained bacterial liquid and spread it to a concentration of On the kanamycin-resistant LB plate, the plate was inverted and cultured at 37°C until monoclonal plaques were formed;

[0041] (2) Induction: Pick four single clones and inoculate them here contain In the LB test tube of kanamycin, shake culture at 250r / min in a shaker at 37°C, when the OD600 reaches 0.1-1.0mM isopropyl-β-D-thiogalactoside was added to three of the test...

Embodiment 3

[0049] Example 3: Comparison of expression levels of recombinant CL7-NgTET1 prokaryotic and mTET1CD eukaryotic series.

[0050] In this embodiment, the expression level of CL7-NgTET1 in E. coli Rossetta strain and the expression level of mTET in 293 cells were analyzed by SDS-PAGE and BCA, and then compared.

[0051] The expression operation of recombinant CL7-NgTET1 in prokaryotic is the same as that in the above-mentioned Example 2. Four groups of samples were taken to run the gel, one group was a blank control, and the other three groups were repeated under 16°C induction conditions, and the supernatant and precipitate were separated for SDS-PAGE.

[0052] The expression operation of GST-mTET in 293 cells is as follows:

[0053] (1) Plasmid and cell preparation: The N-terminal of mTET1CD was fused with GST and constructed into pEE12.4 expression vector to form pEE12.4-GST-mTET1CD plasmid, and the pEE12.4-GST-mTET1CD plasmid was amplified with Escherichia coli DH5α , extra...

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Abstract

The invention provides a CL7 protein capable of shielding nuclease activity. The amino acid sequence of the CL7 protein is shown as SEQ ID NO. 1. The invention also discloses a high-activity recombinant TET enzyme CL7-NgTET1 of which the amino acid sequence is shown as SEQ ID NO: 4, and a prokaryotic expression vector of the high-activity recombinant TET enzyme CL7-NgTET1 of which the structure is pET28A-N6H-MBP-CL7-NgTET1, and also discloses application of the high-activity recombinant TET enzyme CL7-NgTET1 in detection of DNA methylation by an enzyme conversion method. According to the invention, design and transformation are carried out on the basis of NgTET1, and the CL7-NgTET1 recombinant fusion protein is obtained by fusing CL7 domain. According to the recombinant fusion protein, the oxidation capacity of TET protein on 5mC can be remarkably improved, and the in-vitro biological activity of the enzyme is improved. The MBP fusion protein is introduced to the N end of the vector and can be expressed in a prokaryotic system, so that the yield of soluble protein is greatly improved, the problems of low yield and low enzyme activity of TET enzyme are solved, the production cycle is greatly shortened, and the cost is reduced.

Description

technical field [0001] The patent of the invention relates to a CL7 protein, a highly active recombinant TET enzyme CL7-NgTET1, its prokaryotic expression vector and its application, and belongs to the field of biotechnology. Background technique [0002] DNA hydroxymethylase, Ten Eleven Translocase (hereinafter referred to as TET) is a ubiquitous α-ketoglutarate and Fe2+-dependent dioxygenase in eukaryotes, which is highly conserved in the process of biological evolution . TET enzyme is a key protein in the DNA demethylation process, which can convert 5mC into 5caC through a three-step oxidation reaction (5mC-5hmC-5fC-5caC). The existing main TET enzymes include: murine mTET1 / 2, human hTET1 / 2, NgTET1, etc. and their series of mutations, truncations or fusions. [0003] Among them, the recombinant mouse-derived and human-derived TET enzymes with high functional activity are mainly obtained from human 293 series cells, but it is difficult to express them in prokaryotic cell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N9/02C12N15/70C12Q1/25
CPCC12N9/22C12N9/0071C12N15/70C12Q1/25C07K2319/24C07K2319/21C07K2319/50C12N2800/101G01N2333/90245
Inventor 宋东亮韦磊邬升杨李婕刘倩曹振江翱黄开瑜
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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