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System for efficient multi-point interposition of unnatural amino acid into mammalian cells

A non-natural amino acid and mammalian technology, applied in the field of biopharmaceuticals, can solve problems such as difficult direct transfer, bottlenecks in the application of gene codon expansion technology, etc., and achieve the effect of specific modification

Active Publication Date: 2017-08-08
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] 4. Bottleneck in the application of gene codon expansion technology
However, the method of library screening is difficult to directly transfer to mammalian cells

Method used

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  • System for efficient multi-point interposition of unnatural amino acid into mammalian cells
  • System for efficient multi-point interposition of unnatural amino acid into mammalian cells
  • System for efficient multi-point interposition of unnatural amino acid into mammalian cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097] Embodiment 1: the construction of gene carrier

[0098] (1) Construction and acquisition of double reporter plasmids

[0099]The renilla luciferase gene renilla was used as an internal reference, and the peptide GGGGS was used as a linking fragment, which was fused and expressed at the N-terminal of the firefly luciferase gene firefly, and the amber codon UAG was introduced into the linking fragment of the two to obtain a double reporter vector pGL4 -2luc-TAG. Similarly, the amber codon was replaced with an ocher codon (UAA) or an opal codon (UGA), and the dual reporter vectors pGL4-2luc-TAA and pGL4-2luc-TGA were obtained to evaluate the read-through efficiency of the stop codon UAA / UGA, respectively. .

[0100] (2) Construction and acquisition of auxiliary plasmids

[0101] Through the optimization and experimentation of the conditions, it was determined that the tandem expression of the four tRNAs from the 7sk promoter was optimal. The tRNA synthetase (pheRS), f...

Embodiment 2

[0116] Example 2: Expression and Identification of Site-Directed Mutagenesis of GFP / E2

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Abstract

The invention provides a system for efficient multi-point interposition of unnatural amino acid into mammalian cells. The system comprises an expression vector for expressing protein containing unnatural amino acid, wherein the unnatural amino acid is encoded by a codon UAG and contains a vector of methanogen tRNA (tRNAPyl) and pyrrolysyl-tRNA synthetase (PylRS); and the system also comprises an eRF1 cell line obtained from mutation. In addition, the invention also provides a method for preparing fixed-point mutant protein with the application of the system.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a system for efficiently multi-point insertion of unnatural amino acids in mammalian cells and a method for efficient multi-point insertion of unnatural amino acids for expressing proteins (such as antibodies and glycosylated proteins) in mammalian cells. Background technique [0002] 1. Gene codon extension technology [0003] Proteins undertake highly diverse functions in organisms, including maintaining the stability of cell structures, catalyzing chemical reactions in vivo, regulating cellular responses through signal transduction, targeting foreign molecules through the immune system, and so on. Proteins only use 20 kinds of natural amino acids to perform a series of significant functions, and the only 20 kinds of natural amino acids carry a limited number of functional groups, which cannot meet the needs of chemical and biological science research on protein structure and fu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/11C12N15/113C12N5/10C07K14/18C12N15/51
CPCC07K14/1833C12N5/16C12N15/11C12N15/113C12N15/85C12N2770/24222C12N2800/107C12N2830/008
Inventor 周德敏夏青徐欢张子威吴一鸣王天畅王妍张礼和
Owner PEKING UNIV
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