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DNA sequence with multi-tag series connection and application of DNA sequence to protein expression and purification system

A sequence and coding sequence technology, applied in recombinant DNA technology, peptides containing affinity tags, peptides containing His tags, etc., can solve the problem of high time and cost, few applications of expression and purification, and no multi-tag tandem elements. And other issues

Active Publication Date: 2019-11-05
KANGMA SHANGHAI BIOTECH LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The DNA templates used for in vitro synthesis usually do not have multi-tag tandem elements, and the time and cost of designing different tags and purification methods for different proteins are relatively high[5]
At present, the application of eukaryotic multi-tag tandem elements to initiate protein expression and purification in vitro is still relatively small

Method used

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  • DNA sequence with multi-tag series connection and application of DNA sequence to protein expression and purification system
  • DNA sequence with multi-tag series connection and application of DNA sequence to protein expression and purification system
  • DNA sequence with multi-tag series connection and application of DNA sequence to protein expression and purification system

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preparation example Construction

[0137] In the present invention, the preparation method of the yeast cell extract is not limited, and a preferred preparation method includes the following steps:

[0138] (i) providing yeast cells;

[0139] (ii) washing the yeast cells to obtain washed yeast cells;

[0140](iii) subjecting the washed yeast cells to destructive treatment to obtain crude yeast extract;

[0141] (iv) performing solid-liquid separation on the crude yeast extract to obtain the liquid part, which is the yeast cell extract.

[0142] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0143] In a preferred embodiment, said centrifugation is performed in a liquid state.

[0144] In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000g, preferably 8000-30000g.

[0145] In the present invention, the centrifugation time is not particul...

Embodiment 1

[0206] Embodiment 1: Eukaryotic cell changes the coding sequence of protein initial translation efficiency and protein expression purification sequence

[0207] 1.1 Find the relevant nucleic acid sequences in the gene bank (Table 1). The STN1 sequence is the Streptavidin gene sequence, and the STN2 sequence is part of the bases modified by synonymous codon substitutions based on the full sequence characteristics without changing the amino acid sequence. In Table 1, the coding sequence marked with Tag I is the coding sequence that changes the translation efficiency of the protein, the one marked with Tag II is the binding sequence for protein expression and purification, and the one marked with Tag III is the connecting sequence.

[0208] Table 1 Related Nucleic Acid Sequences in Multi-Tag

[0209]

[0210]

Embodiment 2

[0211] Embodiment 2: Contain the construction of the in vitro protein synthesis system plasmid of eukaryotic cell translation control sequence and protein expression purification sequence

[0212] 2.1 Whole gene synthesis: connect STN1, STN2 nucleic acid sequence, leader peptide sequence and ubiquitin sequence in series, and use the method of whole gene synthesis to synthesize. The His tag sequence is directly amplified on the template using two pairs of primers.

[0213] 2.2 Construction of plasmid: The plasmid template was constructed as pD2P plasmid, and the translation control sequence and protein expression purification sequence were inserted between the translation initiation codon ATG and eGFP. Different translation regulation sequences and protein expression and purification sequences can be combined, and the specific experimental combinations are shown in Table 2.

[0214] The specific construction process is as follows:

[0215] Use pD2P as a template for plasmid c...

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Abstract

The invention provides a DNA sequence with multi-tag series connection and application of the DNA sequence to a protein expression and purification system and particularly relates to a nucleic acid structure. The nucleic acid structure is provided with a structure from 5' to 3' in the formula I, wherein the structure is Z1-Z2-Z3-Z4-Z5 (I); in the formula, Z1 to Z5 are elements used for constituting the nucleic acid structure; all '-' parts are bond or nucleotide connecting sequences; Z1 is the coding sequence of leading peptide; Z2 is the coding sequence without ubiquitin or with ubiquitin; Z3is the coding sequence of tab proteins; Z4 is the connecting sequence; and Z5 is the coding sequence without foreign proteins or with the foreign proteins. According to the nucleic acid structure, the efficiency of the foreign protein synthesis can be significantly improved, and the expression and purification processes of the target foreign proteins are simplified.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a DNA sequence with multiple tags in series and its application in a protein expression and purification system. 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 [1]. 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 [1]. In the field of biomedicine, protein antibodies, as targeted drugs, are an important means of treating cancer and other diseases[1,2]. [0003] Some translation-enhancing coding sequences and affinity sequences for protein expression and purification have been found in cells from yeast to human, but the research on thes...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N5/10C07K19/00C07K7/08
CPCC07K7/08C07K14/00C07K2319/20C07K2319/21C07K2319/23C07K2319/24
Inventor 郭敏徐开章小铃周子鉴江丹李海洋于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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