Preparation for protein or peptide labeled by unnatural amino acid

An unnatural amino acid and amino acid technology, applied in the field of biopharmaceuticals, can solve the problems of complex separation and purification process, uneven modification of interferon, etc., achieve the effect of optimizing modification conditions, minimizing drug effect reduction, and realizing immunogenicity

Pending Publication Date: 2014-10-15
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] Facing the problems of uneven modification of interferon and complicated separation and purification process in the prior art, there is an urgent need in this field for methods that can specifically modify functional groups at any site.

Method used

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  • Preparation for protein or peptide labeled by unnatural amino acid
  • Preparation for protein or peptide labeled by unnatural amino acid
  • Preparation for protein or peptide labeled by unnatural amino acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Embodiment 1: the construction of the gene vector comprising the human interferon of site-directed mutation

[0107] (1) Acquisition of helper plasmids

[0108] From the General Microbiology Center of China Microbiological Culture Collection Management Committee (Crew preservation address: No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the preservation date is April 8, 2013, and the preservation number is CGMCC No: Plasmid pSUPAR-YAV-tRNA / PylRS (hereinafter referred to as The plasmid is an auxiliary plasmid), which can express tRNA and tRNA synthetase that specifically recognize the unnatural amino acid Lys-azido.

[0109] (2) Obtaining a plasmid containing natural human interferon

[0110] The interferon gene (SEQ ID NO: 2) was obtained through whole gene synthesis. Then connect it to the pET-21a(+) expression vector to obtain the natural interferon expression plasmid (pET21a(+)-IFN(WT).

[0111] (3) Sel...

Embodiment 2

[0121] Example 2: Expression and purification of interferon with site-directed mutation

[0122] In the present invention, constructing pSUPAR-YAV-tRNA / PylRS plasmid and expressing tRNA (tRNA) derived from ancient Methanococcus Pyl ) and pyrrolysyl-tRNA synthetase (tRNA Pyl ) plasmid co-expression, in the host bacteria, in principle, the use of this protein translation system can make the unnatural amino acid Lys-azido incorporated into the protein, thereby causing site-directed mutation of interferon.

[0123] Next, the inventors examined the incorporation possibility of Lys-azido and the production performance of the mutant protein.

[0124] 1: Synthesis and identification of unnatural amino acid Lys-azido

[0125] The chemical synthesis reaction formula of unnatural amino acid Lys-azido is as follows

[0126]

[0127] As described in the above formula, dissolve 2.3 mL of raw material 1 (2-bromoethanol) in a mixed solution of 90 mL of acetone and 15 mL of water, add 3....

Embodiment 3

[0139] Example 3: Polyethylene glycol site-directed coupling of mutants

[0140] When Lys-azido is introduced into interferon, PEG coupling is required by Click reaction.

[0141] 1. Site-directed PEG conjugation of Lys-azido site-directed muteins (see Figure 4 )

[0142] A: Synthesis of PEG-DIBO (methoxy polyethylene glycol amine), for example, refer to Mbua, N.E., Guo, J., Wolfert, M.A., Steet, R., Boons, G.J. Strain-Promoted Alkyne-Azide Cycloditions ( SPAAC) Reveal New Features of Glycoconjugate Biosynthesis.ChemBioChem.2011,12,1912-1921.

[0143] The copper-free catalyzed Click reaction needs to be realized by the ring tension of cyclooctyne, and the modifier is coupled with DIBO (a compound with a cyclooctyne structure) to perform a copper-free catalyzed Click reaction with an azide-containing group.

[0144] B: Coupling of PEG via Cu-free Catalyzed Click Reaction

[0145] The PEG Click reaction system is as follows:

[0146] Lys-azido-IFN-A74 (prepared in Example...

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Abstract

The invention relates to production and preparation for protein or peptide labeled by an unnatural amino acid. For example, the invention relates to human interferon alpha2b subjected to site directed mutagenesis and human interferon alpha2b subjected to site directed modification, and the interferon can be different species or different types. The invention also relates to a method for site directed mutagenesis and site directed modification of interferon. The method comprises using a gene codon expansion technology to introduce an unnatural amino acid to the interferon gene in a site directed way, and having the aid of an unnatural amino acid or a modification agent to realize site directed linkage between growth hormone, and, for example, polyethylene glycol. The invention further relates to application of interferon subjected to site directed mutagenesis or modification, such as application thereof as a stable long-acting interferon, and the like.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a method for site-specific insertion and modification of non-natural amino acids in proteins or peptides (such as interferon). The method includes site-directed introduction of unnatural amino acids into proteins or peptides (such as interferon) based on gene codon extension technology, and site-specific linking of unnatural amino acids with modifiers such as polyethylene glycol. Background technique [0002] The background technology of the present invention is described below by taking interferon as an example, but the following content cannot be understood as an acknowledgment of the prior art in any case, nor can it be considered that the present invention is only applicable to interferon. [0003] (1) Interferon [0004] Interferon is a protein produced by cells under the action of inducers, which has the functions of inhibiting virus replication, inhibiting cell division, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N1/21C12N15/10C07K1/107C07K14/555C12N15/20A61K38/21A61K47/48A61P31/12A61P35/00A61P37/02C12R1/19
Inventor 周德敏陈景贤张博俞飞张传领司龙龙
Owner PEKING UNIV
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