Application of nucleic acid construct containing streptavidin elements to protein expression and purification

A technology for nucleic acid constructs and constructs, which is applied in the biological field and can solve problems such as high time and cost, and lack of Streptavidin components

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

AI Technical Summary

Problems solved by technology

[0006] However, the DNA templates used for in vitro synthesis usually do not have Streptavidin elements, and the time and cost of designing different tags and purification methods for different proteins are relatively high (10)
At present, the application of eukaryotic Streptavidin components to initiate protein expression and purification in vitro is still relatively small

Method used

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  • Application of nucleic acid construct containing streptavidin elements to protein expression and purification
  • Application of nucleic acid construct containing streptavidin elements to protein expression and purification
  • Application of nucleic acid construct containing streptavidin elements to protein expression and purification

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preparation example Construction

[0112] In the present invention, the preparation method of the cell extract is not limited, and a preferred preparation method includes the following steps:

[0113] (i) providing cells;

[0114] (ii) washing the cells to obtain washed cells;

[0115] (iii) subjecting the washed cells to destructive treatment to obtain a crude cell extract;

[0116] (iv) performing solid-liquid separation on the crude cell extract to obtain the liquid part, which is the cell extract.

[0117] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0118] In a preferred embodiment, said centrifugation is performed in a liquid state.

[0119] In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000g, preferably 8000-30000g.

[0120] In the present invention, the centrifugation time is not particularly limited, and a preferred centr...

Embodiment 1

[0172] Embodiment 1 Eukaryotic Streptavidin Amino Acid Sequence Determination

[0173] 1.1 Determination of the amino acid sequence of Streptavidin: find out the relevant nucleic acid sequence and corresponding amino acid sequence from the gene bank, and use it as the corresponding biotin sequence. The WtStreptavidin sequence (STN1) is a natural streptavidin sequence directly transferred from the gene bank. OurStreptavidin sequence (STN2) modified part of the bases by using synonymous codon substitutions according to the characteristics of the entire sequence without changing the amino acid sequence. OurStreptavidin and WildtypeStreptavidin have the same amino acid sequence. The sequence determination of Linker depends on the amino acid sequence being as similar as possible to the natural protein, the homology of the corresponding amino acid sequence is not less than 30%, and the nucleic acid sequence does not contain strong secondary structure sequences, hairpin structures, G-q...

Embodiment 2

[0178] Embodiment 2 Contains the construction of the in vitro protein synthesis system plasmid of eukaryotic Streptavidin nucleic acid sequence

[0179] 2.1 Whole gene synthesis: two artificially designed STN nucleic acid sequences (STN1, STN2) are connected in series and synthesized using the method of whole gene synthesis.

[0180] 2.2 Plasmid construction: For the two artificially designed STN nucleic acid sequences (STN1, STN2), use two pairs of primers, the primers with the suffixes 1f and 1b respectively amplify the corresponding STN fragments from the synthetic DNA fragments, the suffixes are 2f and 2b The primers were used for PCR amplification of pD2P-eGFP plasmid. In the final constructed plasmid, two STN nucleic acid sequences (STN1, STN2) were inserted between the AUG start codon of the pD2P-eGFP plasmid and eGFP. The names of the plasmids are: pD2P-STN2_eGFP and pD2P-STN1_eGFP, respectively.

[0181] Insert the Linker2 sequence into the existing plasmid pD2P-STN...

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Abstract

The invention provides application of a nucleic acid construct containing streptavidin elements to protein expression and purification, and particularly provides the nucleic acid construct. The nucleic acid construct has a formula I structure from 5' to 3', and the formula I is as follows: Z1-Z2-Z3 (I); and in the formula I, Z1, Z2 and Z3 are the elements used for constituting the construct correspondingly, all '-' are bonds or nucleotide connection sequences independently, Z1 represents a coding sequence of a tag protein, Z2 represents a connection sequence, and Z3 represents a coding sequence of a passive protein or a foreign protein. By applying the nucleic acid construct to a protein synthesis system (especially an in-vitro protein synthesis system), expression and purification of theforeign protein can be completed, and an RFU value of the synthesized foreign protein is increased.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of a nucleic acid construct containing a streptavidin element in protein expression and purification. 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 (1). In the field of biomedicine, protein antibodies, as targeted drugs, are an important means of treating diseases such as cancer (1,2). [0003] Yeast expression system, as a new exogenous protein expression system, is being widely used in the field of genetic engineering due to the advantages of both prokaryotic an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K19/00
CPCC07K14/00C07K2319/20
Inventor 郭敏徐开章小铃姜灵轩王海鹏杨宁于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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