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Protein synthesis efficiency enhancing RNA element

A protein synthesis and component technology, applied in the biological field, can solve problems such as less translation

Inactive Publication Date: 2019-03-05
KANGMA SHANGHAI BIOTECH LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are still relatively few eukaryotic endogenous IRESs used to initiate protein translation in vitro.

Method used

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  • Protein synthesis efficiency enhancing RNA element
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Examples

Experimental program
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Effect test

preparation example Construction

[0077] In the present invention, the preparation method of the yeast cell extract is not limited, and a preferred preparation method includes the following steps:

[0078] (i) providing yeast cells;

[0079] (ii) washing the yeast cells to obtain washed yeast cells;

[0080] (iii) subjecting the washed yeast cells to destructive treatment to obtain crude yeast extract;

[0081] (iv) performing solid-liquid separation on the crude yeast extract to obtain the liquid part, which is the yeast cell extract.

[0082] In the present invention, the solid-liquid separation method is not particularly limited, and a preferred method is centrifugation.

[0083] In a preferred embodiment, said centrifugation is performed in a liquid state.

[0084] In the present invention, the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5000-100000×g, preferably 8000-30000×g.

[0085] In the present invention, the centrifugation time is not parti...

Embodiment 1

[0144] Example 1: Determination of the endogenous IRESs sequence of eukaryotic cells

[0145] 1.1 Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) Determination of the sequence of endogenous IRESs: find out the 60 bases upstream of the start codon (ATG) of 9 related genes (Table 1) in the Saccharomyces cerevisiae genome, and name them as the sequences of the corresponding IRESs are ScYMR181c, ScGPR1, ScBOI1, ScFLO8, ScNCE102, ScMSN1, ScGIC1, SceIF4G2 and ScPab1.

[0146] 1.2 Kluyveromyces lactis ( Kluyveromyces lactis ) Determination of the sequence of endogenous IRESs: use Blast to find the homologous genes of 9 genes in 1.1 from the K. lactis genome (Table 1), and then determine the 60 upstream of the start codon (ATG) bases, and as the corresponding IRESs sequences, named KlYMR181c, KlGPR1, KlBOI1, KlFLO8, KlNCE102, KlMSN1, KlGIC1, KleIF4G, and KlPab1, respectively. Since only one homologous gene of SceIF4G2 was found in the K. lactis genome, only 60 bases upstrea...

Embodiment 2

[0149] Embodiment 2: Contain the construction of the in vitro protein synthesis system plasmid of eukaryotic cell endogenous IRESs

[0150] 2.1 Whole-gene synthesis: The sequences of nine IRESs derived from Kluyveromyces lactis were concatenated and synthesized using the method of whole-gene synthesis.

[0151] 2.2 Construction of the plasmid: using a pair of long primers for the 9 IRESs (ScIRESs) derived from Saccharomyces cerevisiae, the sequence of the IRESs was inserted into the existing plasmid Omega-Fluc (SEQ ID NO. 19) by PCR to replace the Omega sequence, respectively The plasmids containing Saccharomyces cerevisiae IRESs were constructed, and their names and sequence numbers are listed in Table 2.

[0152] For the 9 IRESs (KlIRESs) derived from Kluyveromyces lactis, two pairs of primers were used, the primers with the suffixes 1f and 1b were used to amplify the corresponding IRESs fragments from the synthetic DNA fragments respectively, and the primers with the suffix...

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Abstract

The invention provides a protein synthesis efficiency enhancing RNA element and particularly discloses a nucleic acid structure formed by coding sequences of optional promoters, yeast-derived IRES enhancers (such as ScGPR1, ScFLO8, ScNCE102, ScMSN1, KlFLO8, KlNCE102 and KlMSN1) and heterologous proteins. By application of the nucleic acid structure to a yeast in-vitro protein synthesis system, thesynthesized luciferase activity RLU (relative light unit) is extremely high.

Description

technical field [0001] The present invention relates to the field of biotechnology, and preferably relates to an RNA element for 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 trans...

Claims

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Application Information

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IPC IPC(8): C12N15/81
CPCC12N15/81C12N15/815C12N2840/203
Inventor 郭敏王海鹏柴智刘帅龙于雪
Owner KANGMA SHANGHAI BIOTECH LTD
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