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Method for preparing high-activity antifreeze proteins AFP134

An antifreeze protein with high activity technology, applied in biochemical equipment and methods, chemical instruments and methods, recombinant DNA technology, etc., can solve problems such as harsh natural conditions and scarce insect content

Inactive Publication Date: 2017-11-21
昆明医科大学第二附属医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the harsh natural conditions, the content in insects is scarce, and it is difficult to directly isolate and purify from insects.

Method used

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  • Method for preparing high-activity antifreeze proteins AFP134
  • Method for preparing high-activity antifreeze proteins AFP134
  • Method for preparing high-activity antifreeze proteins AFP134

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0037] 1. Amino Acid Codon Optimization

[0038] The amino acid sequence of Rhagium inquisitor antifreeze protein (GeneBank: HQ540314.1) is as follows:

[0039] MGSS SSGLVPRGSHM CRAVGVDGRAVTDIQGTCHAKATGAGAMASGTSEPGSTSTATA TGRGATARSTSTGRGTATTTATGTASATSNAIGQGTATTTATGSAGGRATGSATTSSSASQPTQTQTITGPGFQTAK SFARNTATTTVTAS

[0040] (The underlined part is the amino acid sequence of antifreeze protein, and the part in bold font is 6×His tag)

[0041] According to the above sequence, the above amino acid sequence is encoded by using Escherichia coli preferred codons to obtain an optimized antifreeze protein coding sequence. And at the 5' of the coding sequence, an NdeI restriction site is added, and a stop codon taa and an XhoI restriction site are added at the 3'. The optimization results are as follows, the part in bold font is the restriction enzyme site, and the lowercase font is the stop codon):

[0042] TGTCGCGCGGTCGGCGTGGATGGTCGTGCGGTTACCGACATCCAGGGCACCTGCCACGCCAAAGCC...

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Abstract

The invention relates to the field of antifreeze proteins, particularly relates to the field of genetic engineering expression antifreeze proteins, and discloses a method for preparing high-activity antifreeze proteins AFP134. The method has the advantages that prokaryotic expression vectors of the efficient antifreeze proteins AFP134 are constructed by the aid of advanced genetic engineering technologies, the high-activity antifreeze proteins AFP134 are successfully expressed in prokaryotic expression systems, and purification processes on the basis of affinity chromatography and molecular sieve technologies are studied and developed.

Description

technical field [0001] The invention relates to the field of antifreeze proteins, in particular to a highly active AFP134 antifreeze protein. Background technique [0002] Antifreeze protein (AFP) was first discovered by DeVries in Antarctic fish and has the function of preventing the formation of ice crystals 1 . Afterwards, the presence of AFP was found in many species, including insects and arthropods 2 . These AFPs have an affinity for ice crystals and can lower the temperature at which ice crystals form without affecting the melting temperature. This phenomenon is called thermal hysteresis (TH). In addition, AFP uses its own ice crystal affinity surface to bind to ice crystals, thereby preventing or inhibiting the growth of ice crystals 3 . AFP can also prevent the recrystallization of ice, thereby protecting cells from mechanical damage caused by ice crystals. AFPs have a wide range of applications, and have potential application value in improving the quality of...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/62C07K19/00C07K1/22C07K1/16
CPCC12N15/70C07K14/43563C07K2319/21C12N15/62C12N2800/22
Inventor 奎翔易晓佳王燕
Owner 昆明医科大学第二附属医院
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