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Polypeptide tag and application thereof in in-vitro protein synthesis

A technology of polypeptide labeling and target protein, which is applied in the field of polypeptide labeling and its application in in vitro protein synthesis. It can solve the problems of interfering with the structure and function of the target protein, reducing the efficiency of protein synthesis, and large molecules, so as to increase the expression of the target protein. , small molecular weight and short polypeptide chain

Pending Publication Date: 2021-06-01
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, most of the tags used for protein fusion are polypeptide chains composed of 20-300 amino acids. The disadvantages are: the chain length is too long, the molecule is too large, and, in most cases, it needs to be removed from the target protein after expression , to prevent it from interfering with the structure and function of the target protein, reducing the efficiency of protein synthesis

Method used

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  • Polypeptide tag and application thereof in in-vitro protein synthesis
  • Polypeptide tag and application thereof in in-vitro protein synthesis
  • Polypeptide tag and application thereof in in-vitro protein synthesis

Examples

Experimental program
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Embodiment approach

[0128] As an embodiment, the source of magnesium ions is a magnesium salt selected from the group consisting of one of magnesium glutamate, magnesium acetate or a combination thereof.

[0129] As an embodiment, the amino acid mixture includes 20 natural amino acids, as well as other unnatural amino acids.

[0130] As an embodiment, the molecular weight of polyethylene glycol is 200-12000Da, preferably 400, 600, 800, 2000, 4000, 8000Da. Measured by weight average molecular weight.

[0131] As an embodiment, the energy supply system is selected from the group consisting of one or a combination of glucose, maltose, trehalose, maltodextrin, starch dextrin, phosphocreatine and phosphokinase; preferably, 320 mM malt Dextrin, 6% trehalose.

[0132] As an embodiment, the cell extract is selected from: eukaryotic cells, yeast cells, Kluyveromyces cells, preferably, Kluyveromyces lactis cells. More preferably, it is a K. lactis cell with T7 RNA polymerase integrated into its genome, ...

Embodiment 1

[0135] Example 1 Determining the sequence of the polypeptide tag

[0136] 1.1 The source and determination of the amino acid sequence of the polypeptide tag: there have been public literature reports, and researchers have confirmed through experiments that the first 11 amino acid residues in the N-terminal half-domain of Dunaliella carbonic anhydrase (dca) (see SEQID for the amino acid sequence of NT11 No.: 9; its DNA sequence: see SEQ ID No.: 18) linked to the N-terminal of foreign protein for fusion expression can increase the translation level of YFP (yellow fluorescent protein) and other proteins in BL21(DE3) E.coli cells (Thi Khoa My Nguyen, et al. The NT11, a novel fusion tag for enhancing protein expression in Escherichia coli. 2019; 103(5):2205–2216.). In this example, the amino acid sequence of NT11 is partially deleted or randomly point-mutated, and then the amino acid sequence that can significantly improve the expression of the foreign protein is screened through e...

Embodiment 2

[0149] Example 2 Plasmid construction of eGFP with N-terminal fusion polypeptide tag

[0150] Plasmid construction: Use a pair of primers to connect the coding gene of the polypeptide tag to the N-terminal coding sequence position of eGFP in the pD2P-eGFP plasmid by using a seamless cloning method. For the gene structure, refer to figure 1 . The names of nine of them are: pD2P-1.07-(001-008) and PC (see Table 1). The sequences of the amplification primers of the nine plasmids are as follows: SEQ ID No.: 19-36.

[0151] The specific construction process is as follows:

[0152] Design a pair of primers according to the seamless cloning technique (see Table 2, wherein, the corresponding forward primer with the suffix of PF, and the corresponding reverse primer with the suffix of PR, were respectively based on the above nine plasmids pD2P-1.07-(001-008 ) and PC as the template for PCR amplification, and 5 μL of the amplified product was identified by 1% agarose electrophoresis;...

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Abstract

The invention provides a polypeptide tag on the first aspect, the amino acid sequence of the polypeptide tag is Xaa1Xaa2, Xaa3PHDYNXaa4Xaa5Xaa6, and in the formula, Xaa1, Xaa2, Xaa3, Xaa4, Xaa5 and Xaa6 are respectively and independently amino acids or none; and the polypeptide tag is used for marking a target protein. On the second aspect, the invention provides a polypeptide fusion protein which comprises the following two structures: (1) any polypeptide tag in the first aspect of the invention, and (2) a target protein connected with the polypeptide tag. The invention also provides an in-vitro cell-free protein synthesis system an and application thereof in in-vitro protein synthesis. The fusion protein is constructed by the polypeptide tag and the target protein, so that the expression quantity of the labeled target protein can be effectively improved under the condition that the polypeptide tag is not cut off.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a polypeptide tag and its application in in vitro protein synthesis. Background technique [0002] Proteins are important molecules in cells and are involved in almost all functions of cells. Proteins vary in sequence and structure, determining their differences in function (1). In cells, proteins can act as enzymes to catalyze various biochemical reactions, act as signaling molecules to coordinate various activities of organisms, support biological forms, store energy, transport molecules, and enable organisms to move (2). In the field of biomedicine, protein antibodies, as targeted drugs, are an important means of treating diseases such as cancer (1,2). [0003] In addition to people's understanding of protein synthesis in cells, protein synthesis can also be carried out outside cells. The in vitro protein synthesis system generally refers to the rapid and e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K19/00C12N15/81C12N15/62
CPCC07K7/06C07K14/43595C12N15/815C12N9/0069C12Y113/12007C07K2319/00C12P21/02C12R2001/645C07K2319/35C12N9/88C12R2001/89C12N15/70C12N15/67C12Y402/01001C12N15/62C12N15/11
Inventor 郭敏章小铃于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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