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Signal peptide related sequences and application thereof in protein synthesis

A signal peptide and sequence technology, applied to signal peptide-related sequences and their application in protein synthesis, can solve problems such as affecting the structure and function of target proteins

Inactive Publication Date: 2020-02-21
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, in the in vitro protein synthesis system, a short polypeptide sequence with a length of less than 30 amino acids is generally inserted into the N-terminal of the target protein to improve the translation efficiency of the target protein, but the insertion of some short peptides will significantly affect the structure and function of the target protein (4 ,14)

Method used

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  • Signal peptide related sequences and application thereof in protein synthesis
  • Signal peptide related sequences and application thereof in protein synthesis
  • Signal peptide related sequences and application thereof in protein synthesis

Examples

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preparation example Construction

[0152] In the present invention, the preparation method of the yeast cell extract is not limited, and a preferred preparation method includes the following steps:

[0153] (i) providing yeast cells;

[0154] (ii) washing the yeast cells to obtain washed yeast cells;

[0155] (iii) performing cell disruption treatment on the washed yeast cells, thereby obtaining crude yeast extract;

[0156] (iv) performing solid-liquid separation on the crude yeast extract to obtain the liquid part, which is the yeast cell extract.

[0157] In the present invention, the solid-liquid separation method is not particularly limited, and is preferably centrifugation.

[0158] In the present invention, the centrifugation conditions are not particularly limited, and the centrifugation conditions are 5000-100000g, preferably 8000-30000g.

[0159] In the present invention, the centrifugation time is not particularly limited, and the centrifugation time is 0.5min-2h, preferably 20min-50min.

[0160]...

Embodiment 1

[0254] Example 1 Determination of eukaryotic cell signal peptide-related sequences

[0255] 1.1 Source and determination of signal peptide-related sequences: Randomly intercept the DNA sequence corresponding to the N-terminus of the constructed foreign protein, and screen the sequence or element that significantly improves the expression of the foreign protein through experiments.

[0256] Specifically, select and synthesize 30 nucleotide fragments with a base length of 36, 54 or other lengths, and modify some bases by means of staggered replacement of synonymous codons to improve the success rate of plasmid construction, wherein Including (but not limited to): reducing the annealing temperature of the sequence in order to reduce the GC content in the sequence related to the signal peptide; or using preferred codons. Dozens of plasmids were constructed. After analysis and screening, 30 of them were tested for their effects on foreign protein expression. The results showed that...

Embodiment 2

[0260] Embodiment 2 Containing the construction of the in vitro protein synthesis system plasmid of signal peptide related sequence

[0261] Plasmid construction: Use 1 pair of primers to amplify the selected 30 signal peptide-related sequences and connecting sequences (the connecting sequences contain TEV restriction sites), and use its original plasmid backbone containing the target protein (eGFP as an example) The corresponding reverse primers were used for amplification. After the amplification is completed, 30 signal peptide-related sequences + connecting sequence fragments are inserted into the N-terminal of the target protein. In the final constructed plasmid, 30 signal peptide-related sequences + linker sequence nucleic acid sequences were inserted between the ATG start codon of the pD2P-eGFP plasmid and eGFP. The names of 13 plasmids are: pD2P-1.0SP-(001-013) (see Table 1).

[0262] The specific construction process is as follows:

[0263] Use 2 pairs of primers to c...

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Abstract

The present invention provides signal peptide related sequences and an application thereof in protein synthesis, and specifically provides a signal peptide with a protein expression improvement effectand a coding sequence thereof, a nucleic acid construct is formed by linking the signal peptide coding sequences with foreign protein coding sequences in an operating manner, the efficiency of foreign protein synthesis can be significantly improved, and the expression and purification process of the target foreign protein is simplified. At the same time, the present invention provides a corresponding vector or a combination of the vector, a genetically engineered cell, and a kit, which can be applied in the protein synthesis.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to signal peptide-related sequences and their application in 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 (1,2). 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 (2). [0003] Signal peptides are short peptides located at the N-terminus of proteins that carry information about protein secretion, and they are widely distributed in all prokaryotes and eukaryotes (3,4). The research on si...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/81C12N15/85C12N1/19C12N5/10
CPCC07K2319/02C12N15/70C12N15/625C07K2319/60C07K7/08
Inventor 郭敏徐开于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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