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Growth hormone with site-specific mutagenesis and site-specific decoration, preparation method and applications of growth hormone

A growth hormone and site-directed mutation technology, applied in the application of growth hormone, site-directed mutation and site-directed modification of growth hormone, and pegylated growth hormone derivatives, can solve the heterogeneity of growth hormone modifiers, growth hormone modifiers issues of inhomogeneity

Active Publication Date: 2012-12-26
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] Facing the problem of heterogeneity of growth hormone modifiers existing in the prior art, there is an urgent need in the art to provide methods for improving the heterogeneity of growth hormone modifiers and homogeneous growth hormone modifiers

Method used

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  • Growth hormone with site-specific mutagenesis and site-specific decoration, preparation method and applications of growth hormone
  • Growth hormone with site-specific mutagenesis and site-specific decoration, preparation method and applications of growth hormone
  • Growth hormone with site-specific mutagenesis and site-specific decoration, preparation method and applications of growth hormone

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Experimental program
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Embodiment 1

[0108] Embodiment 1: Construction of the gene carrier comprising the human growth hormone of site-directed mutation

[0109] (1) Acquisition of helper plasmids

[0110] From the place of preservation: China General Microorganism Culture Collection Management Center Culture Collection Address: Address: No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the preservation date is June 14, 2011, and the preservation number is CGMCC Plasmid pACYC was obtained from Escherichia coli pACYC-tRNA / PylRS containing plasmid pACYC-tRNA / PylRS (gifted by Professor Chen Peng, School of Chemistry, Peking University) No: 4951 taxonomically named Escherichia coli - tRNA / PylRS (hereinafter referred to as the helper plasmid), which can express tRNA and tRNA synthetase that specifically recognize the unnatural amino acids Lys-diazirine and Lys-azido.

[0111] (2) Acquisition of plasmids containing natural human growth hormone

[0112] Amon...

Embodiment 2

[0126] Example 2: Expression and purification of human growth hormone with site-directed mutation

[0127] In the present invention, constructing pACYC-tRNA / PylRS plasmid and expressing tRNA (tRNA) derived from ancient Methanococcus Pyl ) and pyrrolysyl-tRNA synthetase (tRNA Pyl ) after the plasmid co-expression, in the host bacteria, in principle, using this protein translation system can make the unnatural amino acid Lys-diazirine or the azide unnatural amino acid Lys-azido with a structure similar to Lys-diazirine be incorporated into protein, resulting in site-directed mutagenesis of growth hormone. However, whether these two amino acids can be incorporated into proteins, and whether they are suitable for large-scale expression and purification, further experiments are needed to explore.

[0128] Next, the inventors examined the incorporation possibility of two unnatural amino acids, Lys-diazirine and Lys-azido, and the production performance of mutant proteins.

[01...

Embodiment 3

[0182] Example 3: Polyethylene glycol site-directed coupling of mutants

[0183] When Lys-diazirine is introduced into growth hormone, PEG coupling can be performed by photocrosslinking. When Lys-azido is introduced into growth hormone, PEG coupling needs to be performed through Click reaction. The following two couplings are respectively Trials were carried out.

[0184] 1: PEG-directed photocrosslinking of Lys-diazirine site-directed mutants (see Figure 7 )

[0185] A: Synthesis of polyethylene glycol monomethyl ether vinyl ether

[0186] Add a certain amount of polyethylene glycol with the general formula ROH (where R represents CH3O-(CH2CH2O)nCH2CH2) (molecular weight 2kD-100kD) into a certain amount of toluene solution, and add 2-3 times the equivalent of strong base at low temperature ( Such as sodium hydride, etc.), after reacting at room temperature for 1 hour, add 2 times the equivalent of vinyl bromide, react at 60°C for 12 hours, then extract the reaction wi...

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Abstract

The invention relates to growth hormone with site-specific mutagenesis and site-specific decoration. The growth hormone derives from human or other animals. The invention also relates to a method of the growth hormone with site-specific mutagenesis and site-specific decoration. The method comprises the steps that unnatural amino acid fixed points are introduced into genes of the growth hormone by using a genetic code extension technology, and unnatural amino acid and a modifier are utilized, such as the fixed point connection of polyethylene glycol and the growth hormone. The invention further relates to applications of the growth hormone with site-specific mutagenesis and site-specific decoration, such as the applications of stable and long-acting growth hormone.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and specifically relates to site-directed mutation and site-directed modification of growth hormone. The site-directed mutation is to use unnatural amino acids to site-directed mutation of natural amino acids of the growth hormone, and also relates to site-directed modification of the site-directed mutation of the growth hormone , such as pegylated somatotropin derivatives. The present invention also relates to a method for site-directed mutagenesis and site-directed modification of growth hormone, the method comprising using gene codon extension technology to site-directly introduce unnatural amino acids into the growth hormone gene, by means of unnatural amino acids and modifiers, such as polyethylene glycol and growth hormone Hormone-point connection. The present invention further relates to the application of site-directed mutation or modified growth hormone, such as the use as a stable, long...

Claims

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Application Information

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IPC IPC(8): C07K14/61C07K17/08C07K17/10C12N15/18C12N15/63C12N1/21C07K1/107A61K38/27A61K47/48A61P5/06A61P13/12A61P31/18A61P39/00
Inventor 周德敏张传领肖苏龙张雨帆俞飞赵传科陈景贤张礼和
Owner PEKING UNIV
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