N-terminal extension type PTEN subtype PTEN zeta protein as well as coding gene and application thereof

An N-terminal, protein technology, applied to the N-terminal extended PTEN isoform PTENζ protein and its encoding gene and application fields, can solve the problems of low translation initiation ability and the like

Active Publication Date: 2021-06-01
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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  • N-terminal extension type PTEN subtype PTEN zeta protein as well as coding gene and application thereof
  • N-terminal extension type PTEN subtype PTEN zeta protein as well as coding gene and application thereof
  • N-terminal extension type PTEN subtype PTEN zeta protein as well as coding gene and application thereof

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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 extension type PTEN subtype PTEN zeta protein as well as a coding gene and application thereof, and belongs to the technical field of functional proteins. According to the PTEN subtype provided by the invention, a section of 28aa polypeptide is connected to the N end on the basis of PTEN protein, and the polypeptide is a Golgi positioning signal peptide section. Translation of the PTEN[zeta] protein starts from a non-classical AUG translation starting site-ATT948 in a 5'-UTR region, and a palindromic structure at the downstream of the site plays an important role in translation starting. The protein with 431 amino acid sequences, which is obtained by starting translation from ATT948, is named as PTEN[zeta]. PTEN[zeta] is mainly distributed in Golgi apparatus and cell membranes. According to the invention, PTEN[zeta] in Hela cells is specifically knocked out by using CRISPR-Cas9, and a RUSH system is used for verifying that the specific knockout of PTEN[zeta] leads to that the transport speed is obviously increased when endoplasmic reticulum is transported to vesicles of 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 Applications(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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