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Expression system for directly achieving fatty acid modification in protein translation process and application

A technology of expression system and translation process, applied in the field of pharmaceutical protein preparation

Active Publication Date: 2018-11-16
杭州中科理化生物医药技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to fundamentally solve various problems caused by chemically modifying fatty acids after protein translation, the present invention uses biosynthetic methods to directly realize fatty acid modification in the translation process

Method used

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  • Expression system for directly achieving fatty acid modification in protein translation process and application
  • Expression system for directly achieving fatty acid modification in protein translation process and application
  • Expression system for directly achieving fatty acid modification in protein translation process and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1: chemical synthesis contains the hydrochloride or trifluoroacetate of HepoK

[0055]

[0056] α-N-Boc-lysine reacts with heptanoyl chloride under alkaline conditions to generate ε-N-heptanoyl-α-N-Boc-lysine, and removes the Boc protecting group under acidic conditions (TFA or HCl) , the corresponding ε-N-heptanoyl-lysine trifluoroacetate or hydrochloride was prepared and confirmed by nuclear magnetic resonance (NMR) (see Figure 4 ).

Embodiment 2

[0057] Embodiment 2: Construction of aminoacyl tRNA synthetase library

[0058] Genetically encoded unnatural amino acid technology (GEUAA) uses orthogonal tRNA / orthogonal aminoacyl tRNA-synthetase molecule pairs to recognize the amber stop codon (TAG) in living cells, and inserts unnatural amino acids into proteins at specific sites through ribosomes. The method of amino acids, and pyrrolysine is a representative of the principle used in nature. Therefore, the present invention uses the aminoacyl tRNA synthetase (PylRS) of Methanosarcina mazei pyrrolysine (Pyl) as a template, and according to the published crystal structure, adopts the degenerate primer mutation method to target Leu301 and Ala302 in the amino acid binding site. , Leu305, Tyr306, Leu309, Cys348 and other positions for saturation mutation, construct >10 8 library of aminoacyl tRNA synthetases (see Figure 5 ).

Embodiment 3

[0059] Embodiment 3: Screening of aminoacyl tRNA synthetase

[0060] The above aminoacyl tRNA synthetase library was screened using forward and reverse double selection methods,

[0061] 1) Forward screening: co-transfect Escherichia coli DH10β electrocompetent E. coli DH10β electrocompetent with the above synthetase library plasmid and the chloramphenicol resistance gene containing the amber stop codon (TAG) and the plasmid of the green fluorescent protein gene containing the TAG. Culture medium containing chloramphenicol and HepoK hydrochloride or trifluoroacetate (1 mM). If PylRS recognizes natural amino acids or HepoK, TAG can be read through, then the chloramphenicol resistance gene and green fluorescent protein are expressed, and E. coli survives and emits green fluorescence.

[0062] 2) Reverse screening: extract the PylRS plasmid in surviving Escherichia coli, and co-transfect Escherichia coli DH10β electrocompetent with the plasmid of the barnase toxin gene containin...

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Abstract

The invention discloses an expression system for directly achieving fatty acid modification in a protein translation process. The expression system comprises fatty acid type unnatural amino acid epsilon-N-heptanoyl-Lysine (HepoK) hydrochloride or trifluoroacetate, and an orthogonal aminoacyl tRNA synthetase mutant and an expression host which are specific for HepoK. By suing a Genetically EncodingUnnatural Amino Acid (GEUAA) technique, inserting HepoK into a position of an amber termination codon (TAG) code in a target protein at a fixed point, protein fatty acid modification is efficiently,specifically, quantitatively and uniformly achieved in the expression host, and multiple limits such as low in-vitro chemical modification efficiency, poor modification uniformity and hard product separation are thoroughly overcome. The invention further achieves co-translation fatty acid modification on medicinal protein glucagon-like peptide-1 (GLP-1) for a first time, and tests show that biological activity of the peptide is not affected by modification.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, in particular to the preparation of a drug protein. Background technique [0002] With the rapid development of biotechnology, protein peptide drugs have become one of the most active and fastest-growing drugs in the field of pharmaceutical research and development in the 21st century. At present, there are more than 200 protein drugs approved for marketing. In addition to antibodies and Fc fusion proteins, there are also a large part of low molecular weight protein drugs, such as hormones, growth factors, cytokines, coagulation factors, etc. Due to the relatively small molecular size, They are readily cleared from the blood circulation by renal filtration (threshold ~50kDa), coupled with protease degradation and receptor-mediated endocytosis, resulting in generally short blood half-lives (minutes to hours). In order to maintain the effective concentration of the drug effect, the freq...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N9/00C12N15/52C07K14/605
CPCC07K14/605C12N9/00C12N15/63
Inventor 王谦郑峰王磊
Owner 杭州中科理化生物医药技术有限公司