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Expression and preparation method of granulysin

A technology of granullysin and expression cassette, which is applied in the field of expression and preparation of granullysin, and can solve the problems of affecting biological activity, difficulty in renaturation, and low expression efficiency

Inactive Publication Date: 2014-04-09
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently commercialized granulysins only have 15kDa form (such as ProSpec, Abnova and Novus Biologicals), and all contain large tags, which may affect their biological activity
Some researchers overcame this difficulty and tried Escherichia coli, Kluyveromyces lactis, and insect cells to express 15kDa granulysin, and found that only insect cells could express 15kDa granulysin, but the yield was very low (less than 1mg / L)
Most of the 9kDa granulolysin used in the current research comes from the E. coli expression system, and the protein is easy to form inclusion bodies during the expression process. Since there are many disulfide bonds in the protein sequence of the granulolysin, it is relatively difficult to refold in the subsequent renaturation. Difficult; in addition, 9kDa granulysin can also be obtained through in vitro transcription / translation expression system and YT cell degranulation extraction, but the yield is very low
[0003] In summary, the sources of the two forms of granulysin are still very limited, which may be due to the cytotoxic effect of granulysin protein on the host, and some eukaryotic or viral expression systems (such as Saccharomyces cerevisiae, insect cells) are susceptible to Its toxicity restriction leads to poor cell growth and very low expression efficiency

Method used

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  • Expression and preparation method of granulysin
  • Expression and preparation method of granulysin
  • Expression and preparation method of granulysin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0181] Embodiment 1, the construction of GNLY expression vector

[0182] The full length of GNLY is 145 amino acids, including the predicted signal peptide, which is located at amino acid positions 1-22. In killer T cells and NK cells, GNLY is synthesized in the form of 15kDa and then cleaved at the first position to form the form of 9kDa (such as figure 1 A).

[0183] The inventors connected the coding sequence of 9kDa and 15kDa GNLY to the 3' end of the α-Factor signal peptide of Saccharomyces cerevisiae, and only kept the restriction site of kex2 protease, and finally cloned it into pPIC9K yeast through BamH I and EcoR I restriction sites in the expression vector. In this way, methanol induces the expression of AOXI promoter to drive the expression of its downstream proteins. Schematic diagram of the structure of GNLY expression vector figure 1 Shown in B.

Embodiment 2

[0184] The screening of embodiment 2, GNLY expression bacterial strain and shake flask expression condition optimization

[0185] After linearizing pPIC9K-GNLYs-His with SalI, it was electroporated into Pichia pastoris GS115 strain. Nine clones were selected from the YPDG plates where the 9kDa and 15kDa GNLY expression strains were cultivated for induced expression. Western blotting was used to detect the expression of GNLY in the culture supernatant of these clones.

[0186] Taking the expression of 9kDa GNLY clones as an example, most of the clones effectively express 9-kD GNLY (such as figure 2 A), the clone with the highest expression was named GS115 / 9kDa GNLY-His. Western blotting showed that the molecular weight of 9kDa GNLY expressed by yeast was between 10-15kDa, and there were two bands (15% gel) with very similar molecular weights (different degrees of glycosylation modification). The expression level of 15kDa GNLY was significantly higher than that of 9kDa GNLY,...

Embodiment 3

[0188] Embodiment 3, the fermentation expression of GNLY pilot scale

[0189] The present inventors cultivated the expression strain GS115 / 9-kD GNLY-His by fed-batch cultivation (fed-batch cultivation) method, and optimized the method flow. The inventors first used a basal salt medium (pH 5.0) containing 4% (v / v) glycerol to amplify the bacteria in the batch stage (the pH was subsequently adjusted to maintain pH 5.0), and the culture temperature was 30°C. When the DO rises sharply (about 22 hours of cultivation), the batch stage ends, and the wet weight of the bacteria reaches 118.7g / L. Then it enters the conversion period, that is, the glycerin feeding period (feeding amount is: 18.15ml / hr / liter, in the feeding glycerin, each liter of 50% w / v glycerin solution contains 12ml of PTM1, PTM1 is a kind of yeast growth needs Trace element salt solution, the composition is: copper sulfate-5H 2 O 6.0g, sodium iodide 0.08g, manganese sulfate-H 2 O 3.0g, sodium molybdate-2H 2 O 0.2...

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Abstract

The invention relates to expression and a preparation method of granulysin. The inventor adopts pichia pastoris to express granulysin protein or protein fragment for the first time, so the high expression of protein can be realized, and the protein biological activity is well kept. According to the invention, the technical problem that the granulysin protein is difficult to be efficiently expressed in the prior art is solved, and a simple and stable fermentation process is provided for granulysin production.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to the expression and preparation method of granulolysin. Background technique [0002] There are two forms of natural granulysin (Granulysin, GNLY), namely 15kDa form and 9kDa form. The currently commercialized granulysins only have 15kDa forms (such as ProSpec, Abnova and Novus Biologicals), and all of them contain large tags, which may affect their biological activity. Some researchers overcame this difficulty and tried Escherichia coli, Kluyveromyces lactis, and insect cells to express 15kDa granulysin, and found that only insect cells could express 15kDa granulysin, but the yield was very low (less than 1mg / L). Most of the 9kDa granulolysin used in the current research comes from the E. coli expression system, and the protein is easy to form inclusion bodies during the expression process. Since there are many disulfide bonds in the protein sequence of th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/81C12N15/12C07K14/47C07K1/22C07K1/18
Inventor 肖卫华郭雨刚栾淦钟永军李光伟孙洁
Owner UNIV OF SCI & TECH OF CHINA