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Preparation method of affinity chromatography purification tag-free genetic recombinant lamprey Lj-RGD3 protein

A chromatographic purification and gene recombination technology, applied in the field of protein purification, can solve problems such as affinity tag interference, and achieve the effect of high purification rate and simple operation.

Inactive Publication Date: 2014-04-02
LIAONING NORMAL UNIVERSITY
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Problems solved by technology

However, for the structural study of proteins, the application of therapeutic drugs or diagnostic reagents, affinity tags have potential interference
Preparation of high-purity gene recombinant protein rLj-RGD3 without any affinity chromatography purification tag (Tag - ) is a necessary step for its future application in drug research, and there is no information about rLj-RGD3 (Tag - ) A report on the purification process of de-affinity chromatography tagged protein

Method used

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  • Preparation method of affinity chromatography purification tag-free genetic recombinant lamprey Lj-RGD3 protein
  • Preparation method of affinity chromatography purification tag-free genetic recombinant lamprey Lj-RGD3 protein
  • Preparation method of affinity chromatography purification tag-free genetic recombinant lamprey Lj-RGD3 protein

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Embodiment Construction

[0026] Follow the steps below:

[0027] a. Introduce a stop codon at the 3' end of the Lj-RGD3 gene sequence, connect it to the vector with Nde I and Hind III restriction sites, and transform it into the expression bacteria to screen and identify positive transformants. The specific steps are as follows:

[0028] a.1 Extract the pET23b plasmid with the plasmid extraction kit of Baobiology;

[0029] a.2 Using the DNA of Lj-RGD3 as a substrate, use primers P1 and P2 to obtain the target gene through PCR reaction, and then use primers P1 and P2 to react with the pET23b plasmid to connect the DNA fragment of the target gene to the vector pET23b; P2 has Nde I and Hind III restriction sites respectively, and these two restriction sites are also the multiple cloning sites of pET23b, which makes it possible to connect the DNA fragment of the target gene with the vector pET23b; and the stop codon is in The introduction of the 3' end of the Lj-RGD3 gene sequence prevents the expression...

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Abstract

The invention discloses a preparation method of an affinity chromatography purification tag-free genetic recombinant lamprey Lj-RGD3 protein rLj-RGD3(Tag<->). The preparation method has simple processes and a high purification rate. According to the preparation method, a recombinant expression vector pET23b-RGD3(Tag<->) without any purification tags is constructed; the recombinant plasmid is transferred into expression bacteria and a genetic recombinant protein is expressed; and through metal-chelating ion affinity chromatography, cation exchange and dialysis, the lamprey Lj-RGD3 genetic recombinant protein without any affinity chromatography purification tags is prepared, is named as rLj-RGD3(Tag<->) protein and has protein purity greater than or equal to 99%.

Description

technical field [0001] The invention relates to a protein purification method, in particular to a gene recombinant lamprey Lj-RGD3 protein rLj-RGD3 (Tag - ) method of preparation. Background technique [0002] Lj-RGD3 is an RGD toxin protein derived from the oral gland secretion of Japanese lamprey. It consists of 117 amino acid residues. The primary structure of Lj-RGD3 has three RGD motifs and a pair of cysteines. In addition to the typical RGD toxin protein characteristics, it also contains 17 histidines and 17 arginines. It is a basic protein of HRG. Gene Cloning and Expression of Eel Oral Gland RGD Motif Protein" in the Chinese invention patent literature, that is, the protein named Lampetrin3 has anti-angiogenesis, anti-tumor proliferation, anti-thrombosis and other effects. [0003] Traditional protein purification is very complicated, not only need to choose salting out method, molecular sieve chromatography, ion exchange chromatography, affinity chromatography and...

Claims

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Application Information

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IPC IPC(8): C07K14/46C07K1/36C07K1/22C07K1/18C07K1/34
Inventor 李庆伟王继红孙琳王心张安伟吕莉
Owner LIAONING NORMAL UNIVERSITY
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