Tandem DNA elements capable of enhancing protein synthesis efficiency

A component and sub-component technology, applied in the biological field, can solve the problems of lack of access to proteins, no research on two types of translation initiation elements connected in series, low efficiency, etc.

Inactive Publication Date: 2019-06-04
KANGMA SHANGHAI BIOTECH LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the efficiency of translation initiation is relatively low when IRES or Ω sequences are used alone at present, and the purpose of rapid, efficient, an

Method used

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  • Tandem DNA elements capable of enhancing protein synthesis efficiency
  • Tandem DNA elements capable of enhancing protein synthesis efficiency
  • Tandem DNA elements capable of enhancing protein synthesis efficiency

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preparation example Construction

[0140] In the present invention, the preparation method of the eukaryotic cell extract is not limited, and a preferred preparation method includes the following steps:

[0141] (i) providing eukaryotic cells;

[0142] (ii) washing the eukaryotic cells to obtain washed eukaryotic cells;

[0143] (iii) performing a cell-breaking treatment on the washed eukaryotic cells, thereby obtaining a crude eukaryotic cell extract;

[0144] (iv) performing solid-liquid separation on the crude eukaryotic cell extract to obtain the liquid part, which is the eukaryotic cell extract.

[0145] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0146] In a preferred embodiment, said centrifugation is performed in a liquid state.

[0147] In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000g, preferably 8000-30000g.

[0148]...

Embodiment 1

[0252] Embodiment 1: the design of the DNA element of eukaryotic cell endogenous IRESs and omega sequence and Kluyveromyces lactis specific Kozak sequence tandem

[0253] 1.1 Determination of endogenous IRESs in Kluyveromyces lactis and Saccharomyces cerevisiae: four endogenous IRESs of Kluyveromyces lactis and their corresponding IRESs in Saccharomyces cerevisiae (Table 1 ) was able to initiate protein synthesis in vitro, and the relative light unit value of Fluc initiated by six IRESs was higher than that of traditional Ω-sequences (KlFLO8, KlMSN1, KlNCE102, ScFLO8, ScMSN1 and ScNCE102), and the other two were lower than that of Ω-sequences (KlBOI1 and ScBOI1)( figure 2 ). It is confirmed that these 8 IRESs are in series with Ω sequence and Kozak sequence.

[0254] 1.2 Determination of 16 tandem elements: A total of 16 tandem DNA elements (KlFLO8 / KlMSN1 / KlNCE102 / KlBOI1 / ScFLO8 / ScMSN1 / ScNCE102 / ScBOI1-Ω-10A and Ω-KlFLO8 / KlMSN1 / KlNCE102 / KlBOI1 / ScFLO8 / ScMSN1 / ScNCE102 / ScBOI1-...

Embodiment 2

[0259] Embodiment 2: Contain the construction of the in vitro protein synthesis system plasmid of tandem DNA element

[0260] 2.1 Plasmid construction: Eight cell endogenous IRESs were inserted into the Ω-10A-Fluc plasmid, respectively located upstream and downstream of the Ω sequence. The specific primers used are shown in Table 2.

[0261] The specific construction process is as follows:

[0262] For the IRES fragment to be inserted and the Ω-10A-Fluc vector plasmid, two pairs of primers were used for PCR amplification, and 10 μL of the amplification products were mixed; 1 μL of the amplification products was added to 20 μL of the amplification products, and incubated at 37 ° C for 6 h; Add 4 μL of the DpnI-treated product to 50 μL DH5α competent cells, place on ice for 30 minutes, heat shock at 42°C for 45 seconds, place on ice for 3 minutes, add 200 μL LB liquid medium for shaking at 37°C for 4 hours, and spread on the cells containing Amp antibiotics. Cultivate overnight...

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Abstract

The invention provides tandem DNA elements capable of enhancing protein synthesis efficiency. Specifically, a nucleic acid construct is formed by an IRES enhancer (such as ScBOI1, ScFLO8, ScNCE102, ScMSN1, KlFLO8, KlNCE102, KlMSN1, and KlBOI1) derived from eukaryotic cells (such as yeast), an omega sequence, and a yeast-specific Kozak sequence in series. The use of the nucleic acid construct of the present invention in a yeast in-vitro biosynthesis system (such as a yeast in-vitro protein synthesis system) can significantly improve protein synthesis efficiency.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a tandem DNA element capable of enhancing protein synthesis efficiency. 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] In cells, the regulation of protein translation plays an important role in many processes such as response to external stress such as nutrient deficiency, cell development and differentiation. The four processes of protein translati...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N5/10C12N1/19C12N15/85C12N15/81C12N15/67
CPCC12N15/67C12N15/81C12P21/00C12N2800/102C12N2840/203
Inventor 郭敏王海鹏柴智徐开周子鉴章小玲陈鉴冰王静于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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