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Pumpkin protein 2 and preparation method and application thereof

A protein and pumpkin technology applied in the field of biochemistry to achieve the effect of saving cumbersome steps and high purity

Inactive Publication Date: 2012-11-28
FUJIAN INST OF RES ON THE STRUCTURE OF MATTER CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

There are no reports on other ribosome-inactivating proteins in pumpkin fruit

Method used

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  • Pumpkin protein 2 and preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: the preparation technology of pumpkin protein 2:

[0028]Take 500g of pumpkin flesh with skin and seeds removed, add 500ml of pH4.5, 10mM acetic acid buffer, homogenize twice on a homogenizer, then adjust pH to 4.0 with 50% glacial acetic acid, and then heat at 16000 at 4°C × g, centrifuged for 30', collected the supernatant, and the supernatant was applied to the 2.6 × 13cm Sp-Sepharose FastFlow column (the column had been balanced with pH4.5, 20mM acetate buffer), and continued to use pH4.5, After washing out a miscellaneous peak with 20mM acetic acid buffer, continue to wash the column with pH6.5, 20mM phosphate buffer, another miscellaneous peak will be washed out, and then use 0-0.6M NaCl (at pH6.5, 20mM phosphate buffer solution) gradient elution to collect the first peak. Mix the first peak collection solution, adjust the pH4 with 50% glacial acetic acid, add 2 times the volume of pH4.5, 20mM acetic acid buffer, add the sample to the 1.6 × 13cm Sp-...

Embodiment 2

[0029] Embodiment 2: Mono-S analysis of strong cation prepacked column:

[0030] The elution chart of pumpkin protein 2 isolated and purified according to the present invention on the Mono-S HR5 / 5 column (see attached figure): liquid A is pH7.5, 10mM phosphate buffer, liquid B is liquid A+1M NaCl, and the flow rate is 1ml / min, after the Mono-S column is equilibrated with solution A, add sample, wash the column with 3ml of solution A, elute 6ml with a linear gradient of 0-30% solution B, then wash 3ml with 100% solution B, and then equilibrate 5ml with solution A. Pumpkin protein 2 peaked when the eluent reached 6.27ml, that is, when the NaCl concentration was 0.16M.

Embodiment 3

[0031] Example 3: Crystal Growth of Pumpkin Protein 2:

[0032] The pumpkin protein 2 purified by the present invention is concentrated to 14mg / ml, the pool liquid is pH 5.4, 0.1M citric acid buffer pH 6.0-7.8 or 0.1M phosphate buffer, 28% PEG6K is used as a precipitant, and the hanging drop method is used to grow single crystals. The single crystal was collected at a synchrotron radiation source to a set of 1.8 Diffraction data, the crystal belongs to P2 1 2 1 2 1 Crystal form, the unit cell parameter is a=55.853 b=65.507 , c=91.754 .

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Abstract

The invention discloses a Cucurbita moschata protein 2 and a preparation method and application thereof, which relates to the field of biochemistry. The invention discloses a new ribosome inactivated protein extracted from flesh of Cucurbita moschata, namely the Cucurbita moschata protein 2 (type 2 Cucurbita moschata ribosome inactivated protein cucurmosin 2). The molecular weight of the Cucurbita moschata protein 2 is determined as 2, 7183 Da by ESI-MS. The Cucurbita moschata protein 2 is put into phosphate buffer, citric acid buffer and Tris-HCl buffer in advance, and a single crystal for the diffraction of X-ray is produced taking carbowax as a precipitator. Through preliminary analysis, the crystal structure shows that the crystal is a type 1 ribosome inactivated protein with the functions of tumor and virus resistance, in particular human immunodeficiency virus resistance, thereby laying a material foundation for obtaining a protein for medicine use or preparing immunotoxin.

Description

technical field [0001] The invention relates to the field of biochemistry, in particular to the separation and purification of pumpkin protein 2, a new ribosome inactivation protein in biochemistry, crystal growth technology and application thereof. Background technique [0002] Ribosome-Inactivating Protein (RIP for short) extracted from higher plants, RIP is a kind of cytotoxin that can inactivate ribosomes in eukaryotic cells, thereby inhibiting protein synthesis in cells. The mechanism is RNA N-glycosidase or polynucleotide · adenosylglucosidase [EC3.2.2.22] activity. According to the structure of the protein molecule and its gene, RIP can be divided into three types. Type 1 RIP is a single peptide chain protein with RNA N-glycosidase activity. For example, Trichosanthin (Trichosanthin, abbreviated as: TCS), α, β-Momorcharin (α, β-Momorcharin, abbreviation: α, β-MOM) from bitter gourd seeds and luffin a, b (Luffina, b) from loofah seeds. Type 2 RIP is a dipeptide chain...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C07K1/14A61K38/16A61P35/00A61P31/18
Inventor 陈明晃
Owner FUJIAN INST OF RES ON THE STRUCTURE OF MATTER CHINESE ACAD OF SCI
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