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Novel peptide tag and uses thereof

A technology of peptide tags and polynucleotides, applied in the field of epitope labeling systems, can solve the problems of reducing test reliability, hindering protein separation and confirmation, etc.

Inactive Publication Date: 2016-06-01
UNIV IND COOP GRP OF KYUNG HEE UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for the amino acid sequences of the c-myc tag and the FLAG tag, most of the proteins in the cells of organisms known so far contain the same sequence, which may induce a non-specific reaction of the antibody that recognizes the tag, while This non-specific antibody reaction will hinder the separation and confirmation of the specific target protein, so there is a problem that the reliability of the test will be reduced [Ksenija Gasicetal., (2005) Plantmolecularbiologyreporter23:9-16]

Method used

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preparation example Construction

[0041] For example, one example of the present invention provides a method for preparing the above-mentioned fusion protein. A method for preparing a fusion protein according to an example of the present invention includes: culturing a transformant introduced with a polynucleotide encoding a fusion protein to express the fusion protein. In this case, the polynucleotide encoding the fusion protein is preferably introduced into the transformant in the form of a recombinant vector. In addition, the above-mentioned recombinant vector includes: a polynucleotide encoding the above-mentioned peptide tag and a polynucleotide encoding a target protein linked to the above-mentioned peptide tag. Among them, the above-mentioned two polynucleotides are preferably linked by a sequence of a polypeptide linker, wherein the above-mentioned polypeptide linker includes a site that can be cleaved by proteolytic enzymes or the like.

[0042] Furthermore, an example of the present invention provid...

Embodiment 1

[0046] Embodiment 1: Cloning of the BphP gene and the BphN gene of Deinococcus radiodurans

[0047] By the PCR method, the gene (SEQ ID NO: 4) encoding the overall length of the bacterial phytochrome (BphP) of Deinococcus radiodurans (Deinococcus radiodurans) and the polypeptide ( BphN) gene (SEQ ID NO: 5) was cloned. Specifically, using the gene (SEQ ID NO: 4) encoding the overall length of the bacterial phytochrome protein (BphP) of Deinococcus radiodurans as a template, using primers for cloning the BphP gene or primers for cloning the BphN gene, And Ex-Taq (TAKARA) polymerase, carry out the PCR of about 30 cycles, thereby obtain the amplified BphPDNA product and BphNDNA product. Table 1 below shows the primers used to obtain the BphPDNA product and the BphNDNA product. Primers appearing in Table 1 below and all primers described later were prepared by Bioneerco. (KR).

[0048] Table 1

[0049]

[0050]The amplified PCR product was electrophoresed (running) on ​​1%...

Embodiment 2

[0052] Example 2: BphP protein and BphN protein of Deinococcus radiodurans (Deinococcus radiodurans) Express

[0053] After the transformed BL21 competent cells were cultured on LB medium, the grown colony (colony) was inoculated in LB medium containing kanamycin, and then inoculated at 37°C for about 8 hours Cultivate (seed culture). Inoculate the cultured cells in LB medium containing kanamycin and cultivate to OD 600 After the value reached a concentration of 0.6, IPTG (isopropyl-1-thio-β-D-galactopyranoside) was added to a final concentration of 0.5 mM, and protein expression was induced at 25° C. for 6 hours. Next, the cells were centrifuged, the supernatant was removed, and the cell pellet was resuspended with binding buffer (100 mM Tris-Cl, 150 mM NaCl, and 10 mM imidazole, pH 8.0). Then, the resuspended cell mass was sonicated to break the cells, and after centrifugation again, only the supernatant was separated, and Ni + - NTA column (QIAGEN) purified the prote...

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Abstract

There are provided peptide tags derived from bacteriophytochrome (BphP) that is photoreceptor protein of Deinococcus radiodurans, an antibody capable of specifically recognizing the peptide tags, hybridoma cell lines capable of producing the antibody, and uses thereof. The novel peptide tag has advantages in that it has a short length and can remove a non-specific reaction of the conventional c-myc tag and FLAG tag. Therefore, in the case of using the novel peptide tag and antibody thereto, the fusion protein expressed in a recombinant cell can be very effectively detected or purified. In addition, an epitope tagging system including the novel peptide tag and antibody thereto can be applied in various fields such as a determination of an intracellular site, a confirmation of functionality, detection and purification of specific protein, and researches on interaction between proteins.

Description

technical field [0001] The present invention relates to a kind of epitope (epitope) labeling system that is used for the detection or purification of target protein, more specifically, relates to a kind of bacterial phytochrome ( A novel peptide tag of Bacteriophytochrome, BphP), an antibody capable of specifically recognizing the above peptide tag, and a hybridoma cell line capable of producing the above antibody. Moreover, the present invention also relates to a polynucleotide encoding the above-mentioned peptide tag; to a vector or transformant including the above-mentioned polynucleotide; and to a fusion protein including the above-mentioned peptide tag and methods and reagents for preparing, detecting or purifying the above-mentioned fusion protein box. Background technique [0002] Useful proteins or polypeptides can be produced synthetically or can be isolated from natural supplies. However, this method is uneconomical in terms of cost and time, and has disadvantage...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K7/08C07K14/195C12N15/31C12N15/63C07K19/00G01N33/68
CPCC07K14/195C07K1/22C07K14/43595C07K16/12C07K2317/33C07K2317/34C07K2319/00C07K2319/40C07K2319/60C12N9/1088G01N33/573G01N33/58G01N33/68G01N33/6803G01N2333/195G01N2333/43595G01N2333/91177
Inventor 夫盛熙韩胎龙金兑林徐胄源梁胜焕
Owner UNIV IND COOP GRP OF KYUNG HEE UNIV
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