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Method for embedded membrane expression and aquaporins (AqpZ) purification in escherichia coli

An aquaporin and Escherichia coli technology, applied in the field of protein expression and purification, can solve the problems of difficult to meet the structural research and preparation of water recovery devices, and achieve the effect of avoiding toxicity

Inactive Publication Date: 2012-08-22
ZHEJIANG UNIV
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Problems solved by technology

Therefore, it is difficult to meet the requirements of structural studies and fabrication of aquaporin-based water recycling devices

Method used

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  • Method for embedded membrane expression and aquaporins (AqpZ) purification in escherichia coli
  • Method for embedded membrane expression and aquaporins (AqpZ) purification in escherichia coli
  • Method for embedded membrane expression and aquaporins (AqpZ) purification in escherichia coli

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Embodiment Construction

[0032] The method for expressing and purifying aquaporin AqpZ in the membrane of the present invention in Escherichia coli comprises the following steps:

[0033] 1. Amplification of Escherichia coli aquaporin AqpZ-HIS: 2 pairs of primers were designed according to the sequence of Escherichia coli aquaporin gene on NCBI. Eight HIS affinity tags were artificially added to the 3' end of the AqpZ gene by designing primers. The oligonucleotide sequence of the upstream primer of the first primer is as shown in SEQ ID No.1, the oligonucleotide sequence of the downstream primer of the first primer is as shown in SEQ ID No.2, the oligonucleotide sequence of the upstream primer of the second primer The nucleotide sequence is shown in SEQ ID No.3, and the oligonucleotide sequence of the downstream primer of the second primer is shown in SEQ ID No.4. According to the characteristics of the multiple cloning sites of the two vectors pMAL-p2 and pMAL-c5e, add Bam HI and Sal I restrict...

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Abstract

The invention discloses a method for embedded membrane expression and aquaporins (AqpZ) purification in escherichia coli. According to the method, firstly, AqpZ- histidine (HIS) of the escherichia coli is amplified, then, recombination expression vectors and inducible expression engineering bacterial strains are constructed, fusion protein myelin basic protein (MBP)-AqpZ-HIS thalli are obtained through ultrasonic crushing, and finally, the AqpZ is obtained through purification. The method has the advantages that the efficient expression of maltose-binding protein-AqpZ-poly histidine binary tag fusion protein is realized in the escherichia coli, and the problems of insoluble inclusion body forming and host cell toxicity caused by the traditional overexpression AqpZ are solved; and in addition, high-purity AqpZ can be obtained only through two turns of simple affinity chromatography separation on the AqpZ expressed by the method, the operation is simple, and the method can be applied to industrial production.

Description

technical field [0001] The present invention relates to a protein expression and purification method, more specifically, relates to a method for expressing and purifying an aquaporin AqpZ in Escherichia coli by using a maltose-binding protein / polyhistidine binary tag expression system. Background technique [0002] The transmembrane transport of water molecules is one of the fundamental activities of life. The rapid transmembrane transport of water molecules is an important factor for organisms to quickly adapt to changes in osmotic pressure in the extracellular environment. Aquaporins Aquaporins are one of the important members of the important intrinsic proteins. It is a specific transmembrane channel for water molecules to enter and exit cells, so it plays an important role in maintaining the homeostasis of the intracellular environment. Aquaporins are found throughout the biological kingdom, and aquaporins have been identified from prokaryotic bacteria to eukaryotic an...

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K14/245C07K1/22C12R1/19
Inventor 徐志南潘剑峰黄磊蔡谨
Owner ZHEJIANG UNIV
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