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A kind of n-terminal extended type pten subtype ptenζ protein and its coding gene and application

An N-terminal and protein technology, applied to N-terminal extended PTEN subtype PTENζ protein and its coding gene and application field, can solve the problem of low translation initiation ability

Active Publication Date: 2022-05-17
PEKING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The non-ATG initiation codons of the single triplet have lower translation initiation ability than ATG. As mentioned earlier, GTG has the highest translation initiation ability in the Kozak sequence, but it is only 3% to 5% of ATG efficiency (Kozak ,1989)

Method used

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  • A kind of n-terminal extended type pten subtype ptenζ protein and its coding gene and application
  • A kind of n-terminal extended type pten subtype ptenζ protein and its coding gene and application
  • A kind of n-terminal extended type pten subtype ptenζ protein and its coding gene and application

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Experimental program
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Effect test

Embodiment 1

[0058] Translation of PTENζ protein starts at ATT 948 identification method

[0059] (1) Using plasmid expression and mutation analysis to show that the translation of PTENζ starts from ATT 948

[0060] According to the identification of some PTEN subtype proteins such as PTENα, PTENβ and other new subtype proteins of PTEN in the previous research work, in the process of research on PTEN and subtype proteins, a slightly higher molecular weight position (55kDa) than the traditional PTEN protein was found. Bands of unknown proteins (see figure 1 , see [1] PTENα, aPTEN Isoform translated through alternative initiation, regulates mitochondrial function and energy metabolism, Hui Liang, Shiming He, Jingyi Yang, et al. Cell Metabolism, 19, 836-848, 2014, May 6. [2] PTENβ is an alternatively translate isoform of PTEN that regulates rDNA transcription, Hui Liang, Xi Chen, Qi Yin, Danhui Ruan, et al. Nature Communications, 2017.).

[0061]Subsequently, different tumor cell lines we...

Embodiment 2

[0074] Physiological functions of PTENζ

[0075] (1) PTENζ is located in the Golgi apparatus, and its N-terminal 28 amino acid protein sequence is a Golgi apparatus positioning signal

[0076] The function of a protein is closely related to its localization. In order to analyze the function and molecular mechanism of PTENζ, reveal whether PTENζ and traditional PTEN proteins have the same subcellular localization. The nucleotide sequences of PTENζ and traditional PTEN protein were amplified from the cDNA of Hela cells respectively, and the kozak sequence (GCCACCATG) was added upstream of the start codon to enhance expression, and then ligated into GFP carrying the C-terminal by homologous recombination. Label within the pEGFP-N1 vector. At the same time, in order to enhance the expression of this subtype protein, the variable translation initiation codon (ATT 948 ) was mutated to ATG and the start codon ATG of the traditional PTEN protein in the plasmid was mutated to CTC, t...

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Abstract

The invention provides an N-terminal extended PTEN subtype PTENζ protein and its coding gene and application, belonging to the technical field of functional proteins. The PTEN subtype provided by the present invention is a 28aa polypeptide connected to the N-terminus on the basis of the PTEN protein, and the polypeptide is a Golgi localization signal peptide. Translation of PTENζ protein is initiated at a non-canonical AUG translation initiation site in the 5’‑UTR region‑ATT 948 , the palindromic structure downstream of this site plays an important role in translation initiation. by ATT 948 The protein with a sequence of 431 amino acids after initial translation was named PTENζ. PTENζ is mainly distributed in the Golgi apparatus and cell membrane. The present invention uses CRISPR-Cas9 to specifically knock out PTENζ in Hela cells, and uses the RUSH system to confirm that PTENζ-specific knockout leads to a significant increase in the transport speed of vesicles from the endoplasmic reticulum to the Golgi apparatus.

Description

technical field [0001] The invention belongs to the technical field of functional proteins, and in particular relates to an N-terminally extended PTEN subtype PTENζ protein and its coding gene and application. Background technique [0002] Protein translation initiation in eukaryotes is delicately regulated. The currently accepted mechanism is that eukaryotic translation initiation factor (eIF, eukaryotic initiation factor), GTP and Met-tRNAiMet form a ternary complex, and then bind to the ribosomal 40S subunit, while the anticodon CAU of Met-tRNAiMet interacts with 40S The start codon ATG of the mRNA on the subunit is complementary paired to form a 40S priming complex, and then the 60S subunit binds to it, eIF then falls off to form an 80S priming complex, thereby initiating protein translation (Jia, 2005). Although ATG has always been considered as the only translation initiation point in eukaryotes, with the deepening of research, it is found that in addition to ATG as t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/16C12N15/55C12N15/113C12N9/22
CPCC12N9/16C12N15/1137C12N9/22C12N2310/20
Inventor 尹玉新梁会汪菡
Owner PEKING UNIV
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