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Phosphorylation modification LEA (late embryogenesis abundant) protein as well as preparation method and application thereof

A phosphorylation and protein technology, applied in the field of biomedicine, can solve problems such as unclear LEA3 protein, and achieve the effects of protective activity, stability and preservation, and enhanced protection

Active Publication Date: 2014-04-30
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, it is unclear whether LEA3 protein can be modified by phosphorylation and what functions the phosphorylated LEA3 protein has

Method used

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  • Phosphorylation modification LEA (late embryogenesis abundant) protein as well as preparation method and application thereof
  • Phosphorylation modification LEA (late embryogenesis abundant) protein as well as preparation method and application thereof
  • Phosphorylation modification LEA (late embryogenesis abundant) protein as well as preparation method and application thereof

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preparation example Construction

[0019] The embodiment of the present invention provides a method for preparing phosphorylated modified LEA protein, comprising the following steps: cloning the LEA protein gene; constructing a prokaryotic expression vector: connecting the LEA protein gene to the prokaryotic expression vector; expressing to obtain the LEA protein The method comprises: transforming the prokaryotic expression vector into Escherichia coli, inducing LEA gene to express and synthesize LEA protein in Escherichia coli, collecting and purifying the LEA protein; using protein kinase CKII to phosphorylate and modify the LEA protein.

[0020] Wherein, the LEA protein is the soybean PM18B gene, and the phosphorylation modification of the PM18B gene can obviously enhance its protective effect on enzymes.

[0021] The present invention also provides a phosphorylated LEA protein, and the phosphorylated LEA protein is a phosphorylated PM18B protein.

[0022] The present invention also provides an application o...

Embodiment 1

[0024] Example 1 Phosphorylation modification exists in soybean PM18 protein

[0025] Hepes and other buffer solutions and heat treatment methods were used to extract the heat-stable proteins of soybean radicles that did not break through the seed coat after germination. After quantification, the heat-stable protein group was subjected to two-dimensional electrophoresis using two-dimensional electrophoresis technology. The electrophoretic pattern is as follows figure 1 shown. The experimental results showed that PM18 contained 5 isomers with the same molecular weight (Mr=35kDa), but different isoelectric points (PI=6.2, 6.0, 5.8, 5.6 and 5.4).

[0026] Using MALDI-TOF / TOF MS to further identify the theoretical and measured values ​​(m / z[M+H] + ), the results show that the theoretical and measured values ​​of the quasi-molecular ion peak of PM18 Tyr-136 (m / z[M+H] + ) (500.3130Da and 402.2715Da), there is a difference of 98.04Da, which is the molecular weight of a phosphoric a...

Embodiment 2

[0027] Example 2 Preparation of phosphorylated LEA protein

[0028] (1) Cloning of soybean PM18B gene

[0029] Total cDNA was prepared from the radicle of soybean seeds (Bainong No. 6) cultured for 24 hours. Using cDNA as a template and PM18AF and PM18AR as primers, a specific fragment of about 970bp was amplified by PCR, which was consistent with the theoretical value, such as figure 2 shown. The gene fragment was ligated with the pMD18-T (simple) vector, and the ligated product was transformed into Escherichia coli TOP10. The positive recombinant bacteria were sequenced, and the results showed that the cloned soybean PM18B gene was compared with the pGmPM18 gene registered on NCBI (GenBank: AF009953.1), and the results were as follows: image 3 As shown, there is a lack of 21bp at the 712bp to 732bp (including these two bases), and we tentatively named the cloned gene as PM18B gene.

[0030] The primer sequences are as follows:

[0031] PM18AF: 5′-CCGTCATGAGGCATCATCATC...

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Abstract

The invention relates to the field of biological medicine, and discloses a phosphorylation modification LEA (late embryogenesis abundant) protein as well as a preparation method and application thereof. The phosphorylation modification LEA protein is connected with phosphate groups at Ser, Thr and Tyr sites, and the preparation method of the phosphorylation modification LEA protein comprises the steps of cloning an LEA protein gene; constructing a prokaryotic expression vector, and connecting the LEA protein gene to the prokaryotic expression vector; converting the prokaryotic expression vector into Escherichia coli, inducing the expression of the LEA gene in the Escherichia coli to synthesize the LEA protein, and collecting and purifying the LEA protein; performing phosphorylation modification on the LEA protein by using casein kinase. The protective effect of the prepared phosphorylation modification LEA protein on protein products is significantly enhanced, so that the phosphorylation modification LEA protein can be widely applied to preparation of a protective agent of the protein products.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a method for preparing phosphorylated modified LEA protein and its application as a protective agent for preparing protein products. Background technique [0002] The LEA (late embryogenesis abundant) protein family has more than 800 members and exists in more than 200 organisms such as plants, bacteria, yeast, nematodes, fungi and chironomids. It accumulates in a large amount in seeds at the late stage of plant development, which is closely related to the stress resistance of plants. The expression regulation of LEA family genes is different. The expression of some LEA genes is not affected by external factors, while the expression of some LEA genes is induced by adversity stress such as drought, low temperature, and high salinity. [0003] According to the conserved sequences of LEA proteins, they can be divided into 7 groups. Among them, LEA3 protein is most widely distributed in ...

Claims

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Application Information

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IPC IPC(8): C07K14/00C07K1/107C12N9/96C12N9/04
CPCC07K14/415C12N15/70
Inventor 刘昀郑易之石晓英吴佳辉胡莹莹
Owner SHENZHEN UNIV
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