Recombinant polypeptide and its application

A polypeptide and amino acid technology, applied in the direction of peptides, desipeptides, hybrid peptides, etc., can solve the problems of reducing affinity with target receptors, complex and large screening process, affecting drug tissue distribution, etc., to increase the hydrodynamic volume , reduce immunogenicity, and reduce the effect of screening workload

Active Publication Date: 2020-06-09
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

XTEN has a significant negative charge (Schellenberger, Volker, et al. Nature biotechnology 27.12 (2009): 1186-1190.), which is different from the traditional PEG modification technology. There are some disadvantages in the significant negative charge: 1. It affects the drug Tissue distribution; 2. Reduce the affinity with the target receptor and reduce the biological activity; and XTEN is basically composed of non-repetitive motifs, the screening process is complicated and huge

Method used

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  • Recombinant polypeptide and its application
  • Recombinant polypeptide and its application
  • Recombinant polypeptide and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Design and Construction of Short Sequences

[0058] The following examples take the design and construction of a short sequence with a length of 10 amino acids as an example. 3-6 kinds of glycine (G), alanine (A), serine (S), threonine (T), proline (P) and lysine (K) constituting the short sequence were respectively selected type amino acids, and none of the amino acid residues will appear consecutively in the short sequence. For example, three types of amino acids, alanine, serine, and lysine, were selected, and 10 peptides containing 10 amino acids were designed, named sequence library PT01 (named after the acronym PT of Polypeptide Tag, polypeptide library, Polypeptides are named after P), the sequence names of the amino acid and nucleotide sequences and the SEQ ID NOs of these segments are listed in Table 1. For example, four types of amino acids, glycine, serine, proline, and lysine, were selected, and 10 peptides containing 10 amino acids were designe...

Embodiment 2

[0067] Example 2: Construction of a short sequence segment of 20 amino acids in length

[0068] The following examples describe methods for constructing short sequence segments of 20 amino acids in length. As described in Example 1, Example 2 can also design this section from scratch. At the same time, the construction of a codon-optimized gene set encoding a sequence of 20 amino acids can also be described by taking a short sequence containing 10 amino acids as an example. In the first step, the pET28a vector was transformed to remove the BspQ I site on the vector framework region. The transformed vector was named DMT vector. The GFP gene (SEQ NO: 155) (with Nco I, BamH I, BspQ I, EcoR I at the 5' end and a HindIII restriction site at the 3' end) was cloned into the Nco I and HindIII sites of the DMT vector , to obtain the recombinant vector DMT-Nco I-BamH I-BspQ I-EcoR I-GFP-HindIII. The carrier DMT is digested with BspQ I, and a stuffer sequence can be inserted at the B...

Embodiment 3

[0072] Example 3: Construction of a short sequence segment of 18 amino acids in length

[0073] As described in Example 2 below, there are two ways to construct the PT18 sequence segment. First, as described in Example 1, the P18 sequence segment was directly designed and synthesized. Second, as for the screening method described in Example 2, the construction of a codon-optimized gene set encoding a sequence of 18 amino acids can be described by taking 9 amino acid sequences as an example. The DMT vector was constructed by the same method as in Example 2. Digestion of vector DMT with BspQ I allows insertion of a stuffer sequence at the BspQ I site. The polypeptide sequence of 18 amino acids is named P18. Its 18 amino acid sequences have [X]2, where X is a peptide segment containing 9 amino acids, and 6 types of amino acids are selected from glycine, alanine, serine, threonine, proline, and lysine. The 9-amino acid peptide segment is named sequence library PT06, and the se...

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Abstract

The invention relates to a composition of recombinant polypeptides with different lengths based on screening and construction. The invention discloses a stable and non-structure polypeptide molecule without immunogenicity. The polypeptide molecule comprises 3-6 types of amino acids selected from glycine, alanine, serine, threonine, proline and lysine is designed through a complete artificial method. The polypeptide molecule and a bio-active protein are subjected to fusion expression so that the problem that the original biological activate protein has poor solubility, high immunogenicity or a short half life.

Description

technical field [0001] The present invention relates to screening and construction of recombinant polypolypeptides of various lengths and a pharmaceutical composition comprising the recombinant polypolypeptides. Background technique [0002] Bioactive macromolecules are generally unstable polypeptides or proteins, and most of them have short half-lives. In order to maintain a certain curative effect, frequent administration of large doses is required. Long-term repeated injections not only increase the pain of patients but also easily cause a series of side effects . In addition, many bioactive peptides and proteins have only limited solubility, or tend to aggregate during recombinant production, requiring complex dissolution and refolding processes. A variety of chemical polymers can be attached to these proteins to change their properties. Of particular interest are hydrophilic polymers with a flexible conformation and good hydration in aqueous solutions. A commonly use...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/00C07K19/00A61K47/64
CPCC07K14/00C07K2319/31
Inventor 姚文兵尹骏高向东田浤包立晨邵美
Owner CHINA PHARM UNIV
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