Recombinant carrot antifreeze protein gene CaAFP and application thereof

A technology of antifreeze protein and carrot, which is applied in application, genetic engineering, plant genetic improvement, etc. It can solve the problems of difficult analysis and purification of target protein, and achieve the effect of simple extraction process, simple operation, and reduced damage

Inactive Publication Date: 2018-03-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, antifreeze proteins are often not processed correctly in E. coli to form active antifreeze proteins, and the proteins expressed in E. coli exist in the form of inclusion bodies, which is very difficult for subsequent analysis.
And because the protein expressed by Escherichia coli is an intracellular protein, breaking the cell will lead to the release of the intracellular protein, which brings great difficulties to the purification of the target protein

Method used

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  • Recombinant carrot antifreeze protein gene CaAFP and application thereof
  • Recombinant carrot antifreeze protein gene CaAFP and application thereof
  • Recombinant carrot antifreeze protein gene CaAFP and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Construction of genetically engineered strain Pichia pastoris-pPIC9K-CaAFP1 and strain Pichia pastoris-pPIC9K-CaAFP2

[0046] Add restriction enzymes EcoR I and Not I to the two gene fragments with artificially synthesized amino acid sequences of SEQ ID NO.01 and SEQ ID NO.02, respectively, and then connect them with the vector plasmid pMD18 to obtain Recombinant plasmids pMD18-T-antifreeze1 and pMD18-T-antifreeze2.

[0047] The plasmids pMD18-T-antifreeze1 and pMD18-T-antifreeze2 were double digested with restriction enzymes EcoR I and Not I, and then the genes CaAFP1 and CaAFP2 were obtained by gel recovery; then they were ligated to the same restriction using T4 DNA ligase. The plasmid pPIC9K digested with endonuclease EcoR I and Not I was used to construct recombinant expression vectors pPIC9K-CaAFP1 and pPIC9K-CaAFP2, and transform them into E. coli JM109, and select them on the LB solid medium plate containing ampicillin resistance Positive recombinants were...

Embodiment 2

[0049] Example 2 Expression of Shake Flask Level Genes CaAFP1 and CaAFP2

[0050] Using the Pichia pastoris-pPIC9K transformed with the empty pPIC9K vector as the experimental control, single colonies of Pichia pastoris-pPIC9K-CaAFP1, strain Pichia pastoris-pPIC9K-CaAFP2 and Pichia pastoris-pPIC9K were respectively inoculated into 50 mL of BMGY liquid medium. Culture in a 250mL shake flask at a constant temperature of 200rpm and 28℃ shaking to the logarithmic growth phase, namely OD 600 For 3~6. At room temperature, the yeast cells were collected after centrifugation at 3000×g for 5 min. The supernatant was discarded, and the cells were resuspended in a 500 mL shake flask with 100 mL BMMY liquid medium. Then, the culture was continued at 200 rpm and 28°C and the expression was induced with methanol.

[0051] After the induction of expression started, 100% methanol was added to the fermentation broth to maintain induction every 12h until the final concentration of methanol in the ...

Embodiment 3

[0055] Example 3 Expression of CaAFP1 and CaAFP2 genes in a 7L fermentor

[0056] Pichia pastoris-pPIC9K-CaAFP1, Pichia pastoris-pPIC9K-CaAFP2 and Pichia pastoris-pPIC9K single colonies were respectively inoculated into a 250mL shake flask containing 50mL seed medium, and cultured at 30℃, 200rpm on a shaker 20h, as the fermentation tank culture seed liquid.

[0057] The seed liquid was inoculated in a 7L fermentor with an inoculum volume fraction of 10%, and the initial liquid volume was 2.5L. During the fermentation process, the dissolved oxygen concentration is maintained above 10%. The whole fermentation process is divided into three stages: glycerol batch culture, glycerol flow addition (including transient glycerol and methanol mixed addition) and methanol induction culture. First, culture was performed in batches at 28°C and pH 6.0 for 10 hours until the glycerol was exhausted (shown as a sudden increase in DO). In the second stage, the DO-Stat method was used to feed a ba...

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Abstract

The invention discloses a recombinant carrot antifreeze protein gene CaAFP and application thereof and belongs to the technical field of food biology. A complete gene sequence of recombinant carrot antifreeze protein which is expressed in pichia pastoris is successfully synthesized by taking a carrot antifreeze protein amino acid sequence as a template, and is subjected to recombinant expression in the pichia pastoris; large-batch preparation of the antifreeze protein is realized through induced expression and the yield is 0.88g/L; assistance is provided for industrial production of the antifreeze protein.

Description

Technical field [0001] The invention relates to a recombinant carrot antifreeze protein gene CaAFP and its application, and belongs to the field of food biotechnology. Background technique [0002] Carrot antifreeze protein refers to an antifreeze protein with cold-inducing properties in carrots. Its thermal hysteresis activity is low, but it contains multiple hydrophilic ice crystal binding domains and has a strong ability to inhibit ice crystal recrystallization. It has a strong protective effect on carrots from freezing damage. [0003] Carrot antifreeze protein was discovered earlier, its own characteristics have been studied more thoroughly, it has a strong ability to inhibit recrystallization and has a wide range of applications. However, as a kind of plant antifreeze protein, there is still the problem of difficulty in preparation. Relying on the extraction method to obtain antifreeze protein from carrots, the workload is large, the process is complicated, the cost is hig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N1/19C12P21/02A23G9/38A21D2/26C12R1/84
CPCA21D2/264A23G9/38C07K14/415
Inventor 张晖刘玫齐希光吴港城王立钱海峰
Owner JIANGNAN UNIV
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