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Escherichia coli ferroprotein mutant capable of removing heme and recombinant system containing same

A technology of Escherichia coli and heme, applied in the field of genetic engineering, can solve problems such as the influence of ferritin stability, and achieve the effect of improving temperature stability

Active Publication Date: 2016-04-20
埃特尼特(上海)生命科学有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Met52 is considered to be the key amino acid site for the binding of E. coli ferritin to heme, but it has been mutated to alanine and its effect on the stability of ferritin has not been reported so far

Method used

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  • Escherichia coli ferroprotein mutant capable of removing heme and recombinant system containing same
  • Escherichia coli ferroprotein mutant capable of removing heme and recombinant system containing same
  • Escherichia coli ferroprotein mutant capable of removing heme and recombinant system containing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The preparation method of Escherichia coli ferritin mutant gene M52A:

Embodiment 2

[0033] Cloning of Escherichia coli ferritin gene M52A:

[0034] The Escherichia coli ferritin gene in the PCR amplification embodiment 1 with following pair of primers:

[0035] Upstream primer: 5'-ATCGATGAAGCGAAACATGCAGATCG-3'

[0036] Downstream primer: 5'-GCTTTCATGGTATTCCACATCGTTCAG-3'

[0037] PCR reaction system: 1 μL synthetic sequence (obtained in Example 1), 1 μL upstream primer, 1 μL downstream primer, 10 μL 5×PrimeSTARTMBuffer, 32.5 μL ddH 2 O, 4 μL dNTP, 0.5 μL PrimeSTARTM HS DNA polymerase.

[0038] PCR reaction conditions: 95°C for 5 min; 30 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec, and extension at 72°C for 4.5 min; extension at 72°C for 10 min; incubation at 4°C.

Embodiment 3

[0040] Verification of recombinant clone, expression vector pET32Ek / LIC-M52A (gained in Example 2):

[0041] Verify the PCR product (prepared in Example 2) on agarose gel: prepare 10% agarose gel, add nucleic acid dye to a final concentration of 0.5 μg / mL, mix 5 μL DNA sample with 1 μL 6×LoadingBuffer, carefully add the sample Well, add DL10000 or 1kbladderMarker at the same time.

[0042] Set 100-120V constant voltage electrophoresis, and stop electrophoresis when the front of bromophenol blue runs to about 1cm away from the positive end of the gel. like figure 1 As shown, there is an obvious band at 7000bp, which is consistent with the theoretical size of the target fragment, indicating that the gene has been successfully cloned.

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Abstract

The invention discloses an escherichia coli ferroprotein mutant capable of removing heme and a recombinant system containing the escherichia coli ferroprotein mutant. A DNA sequence of the escherichia coli ferroprotein mutant capable of removing the heme is shown in SEQ NO:1. Compared with the prior art, the escherichia coli ferroprotein mutant (M52A mutant) capable of removing the heme has the characteristics of reducing protein stability and being chemically induced to regain protein stability, and a quarternary structure of the mutant is not obviously changed and is still a spherical shell structure; wild type ferroprotein Tm (temperature at which 50% protein is denaturized) is 69.9 DEG C, while Tm of the mutant M52A is only 54.3 DEG C, and Tm of the mutant M52A after being chemically induced with chlorhematin is restored to 67.7 DEG C, so that the mutant has regulated temperature stability and can be taken as a protein nano carrier with the temperature stability controlled through induction by virtue of micromolecules.

Description

technical field [0001] The invention relates to a heme-removed Escherichia coli ferritin mutant and a recombinant system containing it, belonging to the technical field of genetic engineering. Background technique [0002] Iron is one of the essential elements for most biological organisms to carry out metabolism, and it plays an important role in maintaining cell growth and metabolism. Ferritin is an important protein widely present in animal, plant and microbial cells to maintain the balance of iron metabolism. [0003] The structure of E. coli ferritin is spherical and consists of iron core and protein shell. The former exists in the protein shell in the form of iron hydroxide and phosphate, and the latter is a highly symmetrical octahedron composed of 24 subunit monomers. structure. Ferritin has an outer diameter of 12nm and an inner diameter of about 8nm. Due to its unique nanoscale three-dimensional structure, it has become a research hotspot in the design of drug ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/245C12N15/70C12N1/21
Inventor 张瑜周亦琛王飞李迅徐徐
Owner 埃特尼特(上海)生命科学有限公司