Protein with chemical modified groups and preparation method thereof

A chemical modification and protein technology, applied in the field of protein drug modification, can solve problems such as non-specific modification

Active Publication Date: 2017-04-26
戴旭东 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problem of non-specific modification in the field of protein and polypeptide drug modification and to make up for the deficiencies of the existing technology, the present invention provides a technology for using protein trans-splicing technology to modify chemical groups to multiple C-terminals of proteins

Method used

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  • Protein with chemical modified groups and preparation method thereof
  • Protein with chemical modified groups and preparation method thereof
  • Protein with chemical modified groups and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1: Preparation of protein C

[0060] We selected the genetically engineered Ssp GyrB break intein for constructing the C-protein. C-protein belongs to the fusion protein, which in this embodiment contains 6×histidine tag, SUMO protein (SUMO, Small Ubiquitin-like Modifier protein), SG (Ssp GyrB) intein C segment (SGsplit I C , SEQ ID NO:1), and the amino acid sequence SAGC containing only one cysteine ​​(ie the first amino acid sequence, SEQ ID NO:3). In addition, another option for the C-protein is to sequentially contain a 6×histidine tag, thioredoxin (T protein), the C segment of the SG (Ssp GyrB) intein (SGsplit I C , SEQ ID NO:1), and the amino acid sequence SAGC containing only one cysteine ​​(ie the first amino acid sequence, SEQ ID NO:3). Of course, those skilled in the art can understand that other tag proteins include maltose binding protein (MBP protein) and glutathione sulfhydryl transferase (GST).

[0061] Specifically, a 6×histidine tag was us...

Embodiment 2

[0063] Embodiment 2: Preparation and preparation of N-protein

[0064] In order to verify the generality of the method, a variety of protein peptide drugs were regarded as target proteins. These protein peptide drugs include granulocyte colony stimulating factor (Granulocyte Colony-Stimulating Factor, G-CSF), human growth hormone (human Growth Hormone, hGH), human interferon α2b (interferon-α2b, IFNα2b), interleukin-15 (Interleukin 15, ILK-15) and uric acid oxidase (Urate Oxidase, UOX). Various above-mentioned protein polypeptide drugs (ie the second amino acid sequence, the specific sequence can be seen in SEQ ID NO: 6-10 in the sequence listing) are sequentially combined with the N fragment of the intein (SGsplitI N ) (SEQ ID NO:2) and 6×His tag fusion expression, that is, N protein (N drug I N h).

[0065] The nucleotide sequence of each N-protein was cloned into a PET-28 vector, pE-N drug I N H. According to the standard conversion method, the various N drug I N H...

Embodiment 3

[0066] Example 3: PEGylation and purification of C-protein

[0067] The schematic diagram of PEGylation and purification of C-protein is shown in Fig. 1 . First the C-protein (HS (or T) I obtained in Example 1 C -SAGC) dialyzed into labeling buffer (140mM NaCl, 2.7mM KCl, 10mM NaCl 2 HPO 4 , 1.8mM KH 2 PO 4 , pH 7.3), and added tris (2-carboxyethyl) phosphine (TCEP) to a final concentration of 1-10 mM. Add thiol-active polyethylene glycol (PG1-ML-20k, NANOCS), the molar ratio of polyethylene glycol to protein is 3-10:1, and place at room temperature for 2.5-12 hours. After the labeling reaction was completed, dithiothreitol (DTT) was added to a final concentration of 1-10 mM. Due to the C-protein (HS(or T)I C -SAGC) there is only one cysteine, so the PEG with sulfhydryl reactive functional group is modified to C-protein (HS(or T)I C -SAGC) at specific sites that form the C-precursor protein (HS(or T)I C -SAGC-mPEG). Use SDS-PAGE gel to detect polyethylene glycol modi...

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Abstract

The invention relates to a technology for modifying chemical groups to the C-terminal of protein in a fixed-point mode through a protein trans-splicing technology. Concretely, by the utilization of the protein trans-splicing technology, polyethylene glycol (PEG) is modified to the C terminal of a granulocyte colony-stimulating factor (G-CSF), the C terminal f a human growth hormone (hGH), the C terminal of human interferon alpha 2b (IFN alpha 2b), the C terminal of interleukin-15 (ILK-15) and the C terminal of urate oxidase (UOX). The invention provides a method for preparing fixed-point C-end modified medicine. Meanwhile, the invention provides a protein intron sequence for fixed-point modified protein polypeptide drugs.

Description

technical field [0001] The invention relates to the field of protein drug modification. The invention relates to the technique of modifying chemical groups to the protein C-terminus by using protein trans-splicing technique. It also provides the preparation of various pegylated drugs, as well as the preparation method. At the same time, it also provides protein intron sequences for site-directed modification of protein polypeptide drugs. Background technique [0002] Polyethylene glycol (PEG) chemical modification is an effective way to prolong the half-life of protein and polypeptide drugs in vivo. Physicochemical properties such as conformation, electrostatic adsorption, and hydrophilicity and hydrophobicity of proteins modified with polyethylene glycol will change. These physical and chemical changes make PEGylated drug proteins significantly better than ordinary protein peptide drugs. The advantages of PEGylated protein polypeptide drugs are mainly reflected in: incr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/107C07K14/47C12N15/12
Inventor 戴旭东刘相钦
Owner 戴旭东
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