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Soluble recombinant tartary buckwheat metallothionein FtMT with cell-penetrating activity and preparation method thereof

A metallothionein, soluble technology, applied in the field of soluble recombinant buckwheat metallothionein FtMT and its preparation

Active Publication Date: 2020-07-03
RES INST OF AGRO PROD PROCESSING SHANXI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing technology has not been able to solve the problem of preparation of soluble recombinant tartary buckwheat metallothionein (FtMT) with membrane penetrating activity.

Method used

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  • Soluble recombinant tartary buckwheat metallothionein FtMT with cell-penetrating activity and preparation method thereof
  • Soluble recombinant tartary buckwheat metallothionein FtMT with cell-penetrating activity and preparation method thereof
  • Soluble recombinant tartary buckwheat metallothionein FtMT with cell-penetrating activity and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Plasmid vector and transmembrane peptide gene modification: Escherichia coli alkaline phosphatase (phoA) gene promoter is inhibited in the presence of excess phosphate, and protein expression is gradually suppressed under starvation (bacterial growth enters the lag phase) Induction, through the phoA promoter controlled by the phosphate concentration to achieve the purpose of gradual chronic expression, reducing the chance of overloading the bacterial expression system to form inclusion bodies, and slow accumulation also alleviates the toxicity of exogenous proteins that are produced during rapid accumulation , gain expression.

[0028] The signal peptide modified pel B is modified from the signal peptide of pectinase pel B, which has higher secretion efficiency and can guide foreign proteins to enter the periplasm through the membrane, significantly reducing the cytotoxicity and inhibition that affect the normal physiological functions of the host cells. The ...

Embodiment 2

[0033] Example 2: Construction and identification of recombinant expression plasmids: TatM-FtMT was synthesized by chemical synthesis in vitro according to the tartary buckwheat metallothionein FtMT coding region gene and TatM coding sequence. Using TatM-FtMT as template, specific primer: phoA-TatM-FtMT-F: 5′-G CCATGG CCTATGGCAGGAAGAAG-3′ (with NCOI cleavage site); phoA-TatM-FtMT-R: 5′-A GGATCC GCAGTTGCAGGGATTGC-3' (containing BamH I restriction site), PCR amplification.

[0034] The PCR reaction conditions were: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s; annealing at 65°C for 30 s; extension at 72°C for 1 min, 30 cycles. Theoretical size of TatM-FtMT amplified products was 280 bp. The PCR product was recovered after detection and identification by 15g / L agarose gel electrophoresis.

[0035] The PCR product and phoA expression vector were double-digested with Nco I and BamH I, and the digested PCR fragment and phoA plasmid were respectively r...

Embodiment 3

[0037] Example 3: Expression and purification of TatM-FtMT induced by recombinant protein:

[0038] The correct recombinant expression vector identified by sequencing was transformed into competent peptide Escherichia coli BL21 (DE3), and the recombinant engineering strain phoA-TatM-FtMT / BL21 (DE3) was constructed. The engineered bacteria were inoculated in LB medium, shaken at 37°C and 200 r / min overnight for 12 h to prepare seeds. The next day, according to the inoculation amount of 2.5% (v / v), inoculate them into the optimized and improved Neidhardt low phosphorus medium respectively, and the medium components are as follows: 0.25g / L enzyme mother extract, 2.5g / L tryptone, 3g / L L (NH4) 2 SO 4 , 5g / L NaCl, 1g / L MgSO 4 , 4g / L glucose, phosphorus content 0.05mmol / L. Add 500 µmol / LZnSO after 30 min of low phosphorus culture 4 Stabilize protein to prevent self-polymerization, induce culture at 30°C, 180r / min.

[0039] Phosphorus content was measured using a phosphorus dete...

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Abstract

The invention belongs to the technical field of recombinant protein preparation, and provides soluble recombinant tartary buckwheat metallothionein FtMT with cell-penetrating activity and a preparation method thereof. The method comprises the steps of firstly starting with an alkaline phosphate phoA promoter, and constructing a phoA-TatM-FtMT recombinant expression vector recombinant expression plasmid through the guidance of a secretion signal sequence modified pel B and the gene fusion of a cell-penetrating peptide TatM; transferring into escherichia coli BL21, and constructing an engineering strain phoA-TatM-FtMT / BL21; and after low-temperature induced expression of a Neidhardt low-phosphorus culture medium, carrying out separation and purification through a Ni<2+> sepharose TM 6 Fast Flow chromatographic column, and thus obtaining the soluble recombinant tartary buckwheat metallothionein with cell-penetrating activity. The cell-penetrating efficiency can reach 80% or more. The method is simple to operate and low in cost.

Description

technical field [0001] The invention belongs to the technical field of recombinant protein preparation, and in particular relates to a soluble recombinant tartary buckwheat metallothionein FtMT with transmembrane activity and a preparation method thereof. Background technique [0002] MT (Metallothionein, MT) is a kind of thermal compound that widely exists in the biological world, has a low molecular weight (6-7 kDa), is rich in cysteine, lacks aromatic amino acids and histidine, and can bind to various heavy metals. Stabilizes intracellular proteins. As early as 1957, professors Margoshes and Vallee of Harvard University isolated a protein containing zinc and cadmium in the process of studying cadmium accumulation in horse kidneys. In 1960, Kagi and Vallee officially named this special protein as metallothionein for the first time. (Metallothionein). Since then, MT proteins have been found and isolated in various mammalian tissues and organs, higher plants, eukaryotic mi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N15/66
CPCC07K14/825C12N15/70C12N15/66C07K2319/10C07K2319/02Y02A50/30
Inventor 何永吉李云龙卢晓霞李红梅胡俊君程哲郭洪李琪
Owner RES INST OF AGRO PROD PROCESSING SHANXI ACADEMY OF AGRI SCI
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