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Multiple His sequence tag and application of multiple His sequence tag to protein expression and purification

A sequence and polynucleotide technology, which is applied to the polyrecombinant amino acid sequence tag and its application in protein expression and purification, can solve problems such as affecting the translation efficiency of target protein

Pending Publication Date: 2019-12-10
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, in vitro protein synthesis systems generally use the method of inserting some relatively specific elements in front of the target protein for protein expression and purification, but the insertion of some elements will significantly affect or even inhibit the translation efficiency of the target protein[3,4]

Method used

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  • Multiple His sequence tag and application of multiple His sequence tag to protein expression and purification
  • Multiple His sequence tag and application of multiple His sequence tag to protein expression and purification
  • Multiple His sequence tag and application of multiple His sequence tag to protein expression and purification

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preparation example Construction

[0121] In the present invention, the preparation method of the yeast cell extract is not limited, and a preferred preparation method includes the following steps:

[0122] (i) providing yeast cells;

[0123] (ii) washing the yeast cells to obtain washed yeast cells;

[0124] (iii) subjecting the washed yeast cells to destructive treatment to obtain crude yeast extract;

[0125] (iv) performing solid-liquid separation on the crude yeast extract to obtain the liquid part, which is the yeast cell extract.

[0126] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0127] In a preferred embodiment, said centrifugation is performed in a liquid state.

[0128] In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000 g, preferably 8000-30000 g.

[0129] In the present invention, the centrifugation time is not parti...

Embodiment 1

[0183] Example 1 Eukaryotic cell nHis amino acid sequence length and base determination

[0184] 1.1 Determination of the length of the nHis amino acid sequence: Use the same histidine codon CAC to design sequences with histidine numbers of 3, 6, 8, 12, 15, 18, 20, and 24, and at the same time, in order to reduce the number of nHis sequences The GC content reduces the annealing temperature of the sequence, improves the success rate of plasmid construction, and modifies some bases (CAC becomes CAT) by staggered substitution of synonymous codons. The specific sequence information is reflected in Table 1.

[0185] Table 1 Related Nucleic Acid Sequences

[0186]

Embodiment 2

[0187] Embodiment 2: the construction of the in vitro protein synthesis system plasmid containing nHis nucleic acid sequence

[0188] Plasmid construction: For the designed 8 nHis nucleic acid sequences, use 1 pair of primers containing nHis and Linker sequences, and insert them into the N-terminal and C-terminal of the target protein (eGFP), respectively. In the final constructed plasmid, 8 nHis+Linker The nucleic acid sequence is inserted between the AUG start codon and eGFP of the pD2P-eGFP plasmid, and the 8 Linker+nHis nucleic acid sequences are inserted between the eGFP and the stop codon of the pD2P-eGFP plasmid. The names of the plasmids are: pD2P-nHis_eGFP and pD2P-eGFP_nHis, respectively.

[0189] The specific construction process is as follows:

[0190] For the plasmid construction of pD2P-nHis_eGFP and pD2P-eGFP_nHis, use 1 pair of primers to carry out PCR amplification respectively, and take 10 µL of amplification products to mix; add 0.5 µL of Dpn I to 10 µL of ...

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Abstract

The invention provides a multiple His sequence tag and application of the multiple His sequence tag to protein expression and purification. Specifically, a nucleic acid builder is composed of coding sequences of nHis, catenation sequences (including the codon optimized catenation sequence and the un-optimized catenation sequence) and coding sequences of foreign proteins. By applying the nucleic acid builder to a protein synthesis system, the translation efficiency of target proteins can be improved, and the foreign proteins can be expressed and purified.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a multiple recombinant amino acid sequence tag and its application in protein expression and purification. 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 polyhistidine tag sequences have been found in the process of protein translation and expression, but the research on cell endogenous tag sequence tags that can improve translati...

Claims

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Application Information

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IPC IPC(8): C12N15/63C07K19/00
CPCC12N15/63C07K14/00C07K2319/21
Inventor 郭敏章小铃徐开周子鉴王海鹏李海洋于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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