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Preparation of cell-penetrating peptide-target protein compound and method for efficiently introducing cell-penetrating peptide-target protein compound into streptomyces living cells

A technology of penetrating peptides and complexes, applied in the field of microorganisms, can solve problems such as the research and application reports of foreign proteins mediated by penetrating peptides.

Pending Publication Date: 2022-06-03
FUJIAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But at present, there are no related research and application reports on the penetration of membrane-penetrating peptides to mediate exogenous proteins into Streptomyces living cells

Method used

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  • Preparation of cell-penetrating peptide-target protein compound and method for efficiently introducing cell-penetrating peptide-target protein compound into streptomyces living cells
  • Preparation of cell-penetrating peptide-target protein compound and method for efficiently introducing cell-penetrating peptide-target protein compound into streptomyces living cells
  • Preparation of cell-penetrating peptide-target protein compound and method for efficiently introducing cell-penetrating peptide-target protein compound into streptomyces living cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

The preparation method of the cell-penetrating peptide-target protein complex comprises the following steps:

(1) Construction of CPP-mCherry protein expression plasmid pET28a-CPP-mCherry: The fusion expression gene of penetrating peptide, 4 glycine-linked peptides, and fluorescent protein mCherry was constructed in E. coli expression plasmid pET-28a(+), the described The penetrating peptides and their amino acid sequences are Tat (YGRKKRRQRRR), ANTP (RQIKIWFQNRRMKWKK), KFF 3 K (KFFKFFKFFK), R 9 (RRRRRRRRRR), K 9 (KKKKKKKK), KH 9 -BP100 (KHKHKHKHKHKHKHKHKHKKLFKKILKYL).

[0025] (2) The plasmid pET28a-CPP-mCherry was transformed into Escherichia coli by chemical transformation with calcium chloride E.coli BL21(DE3) competent cells.

[0026] (3) The CPP-mCherry recombinant expression strain was inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin (Kanamycin, Kan), activated at 37°C, 220 rpm for 8 h, and the activated strain was 1:100 It was inoculated in 250 ...

Embodiment 2

A method for introducing exogenous proteins suitable for Streptomyces living cells, comprising the following steps:

(1) Treatment of Streptomyces spores: Add 4 mL of PBS solution to the Streptomyces plate, then gently scrape the Streptomyces spores with an inoculation loop, pipette and mix evenly, and place the bacterial solution evenly on the plate. In a centrifuge tube, centrifuge at 4000 rpm for 3 min to collect bacterial cells. After washing twice with PBS, remove excess solution to obtain Streptomyces sporozoites.

[0030] (2) Take the same amount of cells and add Tat-mCherry, ANTP-mCherry, (KFF) 3 K-mCherry, K 9 -mCherry, R 9 -mCherry, (KH) 9 -BP100-mCherry, uniformly diluted to 50 μM in PBS. Take 100 μL of the above dilution, add it to the cells, mix gently, and incubate at 34°C for 1 h in the dark; 50 μM of fluorescent protein mCherry without a transmembrane peptide is used as a control.

[0031] (3) After incubation, centrifuge at 10,000 rpm for 1 min to discard ...

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Abstract

The invention discloses a preparation method of a cell-penetrating peptide-target protein compound and a method for efficiently introducing the cell-penetrating peptide-target protein compound into streptomyces living cells, which can achieve the purpose of efficiently transferring an exogenous target protein to the streptomyces living cells without generating obvious influence on cell activity. The method can help the target foreign protein to efficiently cross complex cell wall and cell membrane barriers of gram-positive bacteria, and is helpful for mediating the foreign protein to efficiently enter streptomyces living cells through the method, so as to carry out living cell level or visualization level research on cell gene expression regulation, metabolite yield monitoring and the like.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to the preparation of a cell-penetrating peptide-target protein complex and a method for efficiently introducing it into Streptomyces living cells. Background technique [0002] Cell penetrating peptide (CPP) is a class of short peptides composed of about 5 to 30 amino acids, which can be used as a living cell material delivery system to carry target proteins, Nucleic acids and nanomaterials penetrate the physiological barrier cell membrane of animal cells, and have little effect on cell activity within a certain concentration. At present, CPP has been widely used as a non-invasive drug delivery tool targeting intracellular molecules. Recent studies have shown that CPP can not only carry oligonucleotides, small interfering RNAs, plasmids, nanomaterials, etc. to overcome animal cell membranes, but also can carry small interfering RNAs or plasmids to higher plants, algae and m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/62C12N15/76C07K19/00C07K1/22
CPCC12N15/70C12N15/76C07K14/43595C12N2800/101C07K2319/10Y02A50/30
Inventor 张怀东李芹黄建忠何柳郭丽君郭兴罗鑫格
Owner FUJIAN NORMAL UNIV
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