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Fusion protein containing RNA binding protein and expression vector matched with fusion protein

A fusion protein and expression vector technology, applied in the field of genetic engineering, can solve problems affecting the synthesis efficiency and yield of target protein, and achieve the effects of improving in vitro protein synthesis capacity, reducing restrictions, and improving efficiency and yield

Active Publication Date: 2020-09-29
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For yeast cells, the original mRNA will continue to use resources to synthesize non-target proteins (ie miscellaneous proteins), affecting the synthesis efficiency and yield of target proteins

Method used

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  • Fusion protein containing RNA binding protein and expression vector matched with fusion protein
  • Fusion protein containing RNA binding protein and expression vector matched with fusion protein
  • Fusion protein containing RNA binding protein and expression vector matched with fusion protein

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0108] In the present invention, the preparation method of the eukaryotic cell extract is not limited, and a preferred preparation method includes the following steps:

[0109] (i) providing eukaryotic cells;

[0110] (ii) washing the eukaryotic cells to obtain washed eukaryotic cells;

[0111] (iii) performing a cell-breaking treatment on the washed eukaryotic cells, thereby obtaining a crude eukaryotic cell extract;

[0112] (iv) performing solid-liquid separation on the crude eukaryotic cell extract to obtain the liquid part, which is the eukaryotic cell extract.

[0113] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0114] In a preferred embodiment, said centrifugation is performed in a liquid state.

[0115] In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000 g, preferably 8000-30000 g.

[011...

Embodiment 1

[0137] Example 1 C-terminal or N-terminal targeted insertion of eIF3a gene into MS2 CP

[0138] the code Kl The nucleotide sequence of eIF3a is shown in SEQ ID NO:1, and its amino acid sequence is shown in SEQ ID NO:2. the connection Kl The nucleotide sequence of the connection sequence between eIF3a and MS2 CP is shown in SEQ ID NO:3, and its amino acid sequence is shown in SEQ ID NO:4. The nucleotide sequence encoding MS2 CP is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6. said Kl The nucleotide sequence of the eIF3a-MS2 CP fusion protein is shown in SEQ ID NO: 7, and its amino acid sequence is shown in SEQ ID NO: 8.

[0139] Select the construction method with or without linker sequence, set Kl Construction of connection between eIF3a and MS2 CP Kl eIF3a-MS2 CP fusion protein.

[0140] Kl sequence determination

[0141] (1) According to the Kl The C-terminal of eIF3a gene was inserted into MS2 CP, the PAM sequence (NGG) was selected, a...

Embodiment 2

[0162] Example 2 C-terminal or N-terminal targeted insertion of eIF3a gene into Qβ CP

[0163] the code Kl The nucleotide sequence of eIF3a is shown in SEQ ID NO:1, and its amino acid sequence is shown in SEQ ID NO:2. The nucleotide sequence encoding QβCP is shown in SEQ ID NO:12, and its amino acid sequence is shown in SEQ ID NO:13. said Kl The nucleotide sequence of the eIF3a-Qβ fusion protein is shown in SEQ ID NO:14, and its amino acid sequence is shown in SEQ ID NO:15.

[0164] Select the construction method with or without linker sequence, set Kl eIF3a is linked to Qβ CP.

[0165] Kl plasmid

[0166] exist Kl The gRNA design for inserting the C-terminus of the eIF3a gene into the Qβ CP is the same as (1) in Example 1.1;

[0167] exist Kl The gRNA design for inserting the N-terminus of the eIF3a gene into the Qβ CP is the same as (2) in Example 1.1.

[0168] Donor DNA plasmid construction and amplification

[0169] The pMD18T-vector, eIF3aC-F1 and eIF3aN-F1 use...

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Abstract

The invention provides a fusion protein containing RNA binding protein and an expression vector matched with the fusion protein. Specifically, the fusion protein provided by the invention can be combined with (or matched with) the corresponding expression vector for use, so that the in-vitro translation expression efficiency can be greatly improved. In addition, compared with common eukaryotic mRNA, mRNA translated by the expression vector provided by the invention does not contain a polyA structure. Furthermore, the invention provides application of the fusion protein containing the RNA binding protein and the expression vector matched with the fusion protein in cell-free protein synthesis.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to fusion proteins containing RNA binding proteins and their corresponding expression vectors, and their application in a cell-free protein synthesis system to improve protein synthesis. Background technique [0002] Proteins are important molecules in cells and are involved in almost all functions of cells. Different sequences and structures of proteins determine their different functions. In cells, proteins can be used as enzymes to catalyze various biochemical reactions, and as signal molecules to coordinate various activities of organisms, to support biological forms, store energy, transport molecules, and make organisms move. In the field of biomedicine, protein antibodies, as targeted drugs, are an important means of treating diseases such as cancer. [0003] The four processes of protein translation include translation initiation, translation elongation, translation termi...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/81C12N1/19C12P21/02
CPCC07K14/005C12N15/815C12N15/81C12P21/02C07K2319/85
Inventor 郭敏王静许乃庆姜灵轩于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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