Fusion protein containing fluorescent protein fragment and application of fusion protein

A fusion protein and fluorescent protein technology, applied in the field of fusion proteins, can solve problems such as difficult separation, strong hydrophobicity, and low specific gravity of the target protein

Active Publication Date: 2020-09-29
NINGBO KUNPENG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently available fusion protein methods for peptide expression present many technical problems, especially for the generation of peptides of less than 50 amino acid residues
For example, the fusion protein in the prior art has a large molecular weight, strong hydrophobicity, difficult to separate, low specific gravity of the target protein, low fusion ratio, stable structure, difficult to digest, and a lot of dead volume on the ion column and hydrophobic column.

Method used

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  • Fusion protein containing fluorescent protein fragment and application of fusion protein
  • Fusion protein containing fluorescent protein fragment and application of fusion protein
  • Fusion protein containing fluorescent protein fragment and application of fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0217] Example 1 Construction of expression vector A1-u4-u5-TEV-R-MiniINS

[0218] The expression construct A1-u4-u5-TEV-R-MiniINS contains the gene encoding human insulin protein fused to the C-terminus of A1-u4-u5. The connecting peptide between A1-u4-u5 and the insulin protein MiniINS is the octapeptide Glu-Asn-Leu-Tyr-Phe-Gln-Gly-Arg. The octapeptide can be hydrolyzed by trypsin at the carboxyl end of Arg, and can also be hydrolyzed by TEV protease between Gln-Gly. The DNA sequence of the octapeptide is codon-optimized, which can realize high-level expression of functional proteins in Escherichia coli.

[0219] The fusion recombinant protein fragments used were all synthesized by GenScript and loaded into the pUC57 vector, and "A1-u4-u5-TEV-R-MiniINS" was synthesized from the synthetic vector "pUC57-A1-u4" using restriction enzymes NcoI and XhoI -u5-TEV-R-MiniINS”, cut the expression vector “pBAD / His A(KanaR)” with restriction endonucleases NcoI and XhoI at the same time...

Embodiment 2

[0220] Example 2 Construction of expression construct A1-u4-u5-TEV-R-GLP1

[0221] Construct and synthesize pUC57-A1-u4-u5-TEV-R-GLP1 as in Example 1, and cut the expression vector "pBAD / His A(KanaR)" with restriction enzymes NcoI and XhoI at the same time, and digest the product Separation by agarose electrophoresis, extraction using an agarose gel DNA recovery kit, and finally linking the two DNA fragments using T4 DNA ligase. The ligated product was chemically transformed into Escherichia coli Top10 cells, and the transformed cells were cultured on LB agar medium containing 50 μg / mL kanamycin (10 g / L yeast peptone, 5 g / L yeast extract powder, 10g / L NaCl, 1.5% agar) overnight. Pick 3 viable colonies and culture overnight in 5 mL liquid LB medium (10 g / L yeast peptone, 5 g / L yeast extract powder, 10 g / L NaCl) containing 50 μg / mL kanamycin, and use plasmid mini-extraction Kit for plasmid extraction. Then, the extracted plasmid was sequenced using the sequencing oligonucleot...

Embodiment 3

[0222] Example 3 Expression, isolation and purification of A1-u4-u5-TEV-R-MiniINS fusion protein

[0223]In order to express the fusion fragment of A1-u4-u5-TEV-R-MiniINS containing the amino acid sequence of SEQ ID NO.:21. Plasmid pBAD-A1-u4-u5-TEV-R-MiniINS and pyrrolysine aminoacyl tRNA synthetase plasmid pEvol-pylRs-pylT (wherein, pyrrolysine aminoacyl tRNA synthetase plasmid pEvol- pylRs-pylT is used to express aminoacyl tRNA synthetase and tRNA, as shown in Example 1 of Patent Application No. 2011103886241) and transformed into E. coli strain Top10 together. The transformation solution was placed on LB agar medium containing 25 μg / mL kanamycin and 17 μg / mL chloramphenicol overnight. Pick a single colony and culture overnight in LB liquid medium containing 25 μg / mL kanamycin and 17 μg / mL chloramphenicol. Then, the overnight culture was inoculated into 100 mL of TB medium containing 25 μg / mL of kanamycin and 17 μg / mL of chloramphenicol (liquid TB medium: 12 g / L yeast pep...

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Abstract

The invention relates to fusion protein containing a fluorescent protein fragment and an application of the fusion protein. Specifically, the invention provides the fusion protein, and the fusion protein has a structure as shown in (P1-L1)s-A1-(X)n-A2-(L2-P2)t. (X)n or A1-(X)n in the fusion protein can promote the folding and expression of fusion target peptide, improve the solubility of the fusion protein and reduce the intermolecular interaction of the fusion protein, so that the fusion target peptide can be folded under the high concentration with commercial significance. (X)n or A1-(X)n inthe fusion protein can be digested into a plurality of oligopeptides of which the lengths are far less than the length of the target peptide, so that the separation of the oligopeptides from the target peptide is better facilitated, and the purification of the target peptide is more convenient.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to fusion proteins containing fluorescent protein fragments and uses thereof. Background technique [0002] Peptides are a class of biomolecules that are widely used in many fields of biomedical research as reagents, as therapeutic drugs in the treatment of diseases, as diagnostic agents in the detection of pathogenic bacteria, and as biomarkers. There are usually two ways to synthesize peptides, one is chemical synthesis, and the other is recombinant expression. Chemical synthesis has been used to prepare a variety of therapeutic peptides, including cortirelin, parathyroid hormone (PTH), glucagon-like peptide (GLP-1) and its derivatives exenatide and liraglu peptide, enfuvirtide, calcitonin, bivalirudin, ziconotide, sermorelin, somatorelin, secretin, teduglutide, and insulin. This method requires multi-step condensation of amino acid fragments to form peptides, and also requires tedi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21
CPCC07K14/62C12N15/70C07K2319/60C12N15/62C07K2319/35C07K2319/50C07K14/605
Inventor 查若鹏吴松张振山刘慧玲陈卫
Owner NINGBO KUNPENG BIOTECH CO LTD
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