Neurored mutant protein and preparation method thereof

A mutant and protein technology, applied in the field of neuroglobin mutant protein and its preparation, can solve the problem of not improving the stability of neuroglobin

Active Publication Date: 2018-11-27
NANHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Neurored mutant protein and preparation method thereof
  • Neurored mutant protein and preparation method thereof
  • Neurored mutant protein and preparation method thereof

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Embodiment 1

[0030] Based on genetic engineering and protein engineering, using site-directed mutagenesis technology, cysteine ​​(Cys) was introduced into the 15th position of neuroglobin, and the mutant plasmid was expressed in E. coli BL21 (DE3), and 176g of bacteria were broken by 60W ultrasonic for 1h, followed by 176g / L, 195g / L ammonium sulfate salting-out dialysis and DEAE anion exchange, column separation G75, cation exchange column MonoQ method, to separate and purify protein, and obtain A15C Mb mutant protein.

[0031] The amino acid sequence (SEQ ID NO.1) of A15C Mb is as follows:

[0032] MERPEPELIRQSWRCVSRSPLEHGTVLFARLFALEPDLLPLFQYNCRQFSSPEDCLSSPEFLDHIRKVMLVIDAAVTNVEDLSSLEEYLASLGRKHRAVGVKLSSFSTVGESLLYMLEKCLGPAFTPATRAAWSQLYGAVVQAMSRGWDGE

Embodiment 2

[0034] The WT Ngb (wild-type neuroglobin) and A15C Ngb proteins were respectively dissolved in a potassium dihydrogen phosphate-potassium hydroxide buffer solution (pH 7.0) so that the concentration of the protein solution was 10 μM. Add a small amount of high-concentration hydrochloric acid solution to the two protein solutions several times to lower the pH value step by step, record the protein UV-visible spectra corresponding to each pH value, and obtain the corresponding full spectrum ( figure 1 ) and the UV absorbance value at 413nm of the soret peak (A 413nm ) changes with the decrease of pH value ( figure 2 ). Through equation fitting, the denaturation midpoint of WT Ngb was pH 3.3, while that of A15C Ngb was pH 2.6, which indicated that the mutant protein A15C was more stable.

Embodiment 3

[0036] Prepare 200 μL of WT Ngb protein and A15C Ngb protein at a concentration of 2 mM, respectively, and add 10 μL of 2 mM WTNgb protein and A15C Ngb protein samples into 2 mL of a certain gradient concentration of guanidine hydrochloride solution (0-6.0 M) and mix well. Under the condition of 25°C, after mixing evenly, let it stand for 30 minutes, then measure the ultraviolet spectrum of the protein under different guanidine hydrochloride concentrations in sequence, and record the change of the whole spectrum ( image 3 ). The obtained spectral data is plotted by Origin software, with different guanidine hydrochloride concentrations as the abscissa, and the protein Soret peak absorbance value as the ordinate, and the Boltzmann equation is used to fit the guanidine hydrochloride concentration at the midpoint of denaturation, and the processing is normalized. Figure ( Figure 4). Through equation fitting, the midpoint of denaturation of WT Ngb corresponds to a concentration...

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Abstract

The invention belongs to the technical field of biology, and discloses a neurored mutant protein and a preparation method thereof. The neurored mutant protein disclosed by the invention is based on genetic engineering and protein engineering technology, and neurored mutant protein A15C Ngb is obtained by introducing cysteine(Cys15) at the site 15 of human neuroglobin, with an amino acid sequence thereof as shown in SEQ ID NO. 1. The neurored mutant protein A15C Ngb prepared according to the invention has significantly improved pH stability, chemical denaturation stability and thermal stabilitycompared with WT Ngb, and can be widely used for studying the structure and function relationship of the human neuroglobin, and other heme protein molecular design based on the human neuroglobin.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a neurored mutant protein and a preparation method thereof. Background technique [0002] Neuroglobin (Neuroglobin, Ngb) is a heme protein first discovered by German scientists in humans and mice in 2000. It is mainly expressed in nerve tissues such as the brain and retina. It consists of a peptide chain containing 151 amino acids and blood red Compared with vertebrate hemoglobin and myoglobin, its amino acid sequence homology is very low (20%-25%). Through sequence analysis, neuroglobin is older than myoglobin. Ngb has a hexacoordinate structure in both low iron and high iron states, and contains a pair of disulfide bonds and a single cysteine ​​120 (Cys120). The physiological functions of Ngb that have been known so far are: ①Oxygen carrier function, Ngb’s high oxygen affinity can reversibly bind oxygen; ②In the case of hypoxia, ischemia and oxidative damage, neuroglobin will be e...

Claims

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Application Information

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IPC IPC(8): C07K14/805C07K1/36C07K1/34C07K1/30C07K1/18C07K1/16C12N15/12C12N15/70G01N33/72
CPCC07K14/805C12N15/70G01N33/721
Inventor 林英武刘海啸高淑琴
Owner NANHUA UNIV
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