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Compositions and methods related to mrna translational enhancer elements

A technology of enhancing factor, R1N1S1GAGM1GR2M2R2R3R4, which can be used in drug combination, DNA preparation, recombinant DNA technology, etc., and can solve the problem that it cannot function as a translation enhancing factor.

Inactive Publication Date: 2011-01-05
THE SCRIPPS RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some translation enhancers (TEEs) can also act as ribosome recruitment points, which can promote the internal initiation of translation; however, the vast majority of internal ribosome entry sites (IRESe) cannot function as translation enhancers (TEEs) the role of

Method used

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  • Compositions and methods related to mrna translational enhancer elements
  • Compositions and methods related to mrna translational enhancer elements
  • Compositions and methods related to mrna translational enhancer elements

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Example 1 Positive Feedback Vector System for Identifying Translation Enhancers

[0101] TEE recognition involved the use of a dual single-actor positive feedback vector: one cistron encoding the Gal4VP16 transcription factor and the other cistron encoding Renilla luciferase. Both cistrons contain a minimal promoter with 4 copies of an upstream activating sequence (UAS), which enhances transcription when bound to Gal4VP16. When introduced into cells, this minimal promoter drives extremely low levels of messenger ribonucleic acid and encoded protein. Those sequences that enhance translation into the 5' primer of the Gal4VP16 cistron drive translation of the Gal4VP16 mRNA. The Gal4VP16 protein can then bind to these two promoters to enhance transcription, driving its own transcription and that of another cistron in the promoters of the two genes through specific binding sites to start a positive feedback loop. TEEs were identified from a random library of nucleotide seq...

Embodiment 2

[0105] Example 2 Identification of Consistent Translational Enhancer Motifs

[0106] Using a dual single-actor positive feedback vector system as described above, many short nucleosides that function as translational enhancers in Chinese hamster ovary (CHO) and other cell lines, including BHK (small hamster kidney) cells Acid sequences are identified. These translational enhancer elements (TEEs) range in length from seven to twelve. Such as image 3 As shown, multiple copies of these TEEs enhanced protein production up to 25-fold in CHO cells. In each example, there were at least 5 identical copies of the TEE linkage in the 5' primer of the firefly luciferase cistron. The first construct is a size comparison control that does not contain TEE. Constructs were briefly transfected into CHO cells and tested 2 days later. Translation efficiency relates to the activity of size versus control constructs. exist image 3 Among them, the signal numbers of the top 13 bars were r...

Embodiment 3

[0110] The activity of the messenger ribonucleic acid translation enhancer factor of embodiment 3 synthesis

[0111] Based on the consensus motifs annotated above, various specific TEE sequences roughly corresponding to motifs 1 and 2 were synthesized and tested for translational enhancing activity (see SEQ ID NO: 5-35 of Table 1). The DNA fragment containing the test sequence is produced by synthesizing and annealing two overlapping, complementary oligonucleotides to each other. Annealed oligonucleotides have unpaired nucleotides at each end, which provide sticky ends for cloning into vectors using EcoR1 and BamH1. This fragment was cloned into the 5' primer of the luciferase reporter mRNA in a positive feedback expression vector as described in Example 1. The Renilla luciferase construct containing the motif sequence was then transiently transfected into CHO cells. Cells were assayed 3 days later for the ability of these test sequences to enhance translation. A comparat...

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Abstract

Provided are mRNA translational enhancer elements (TEEs), e.g., SEQ ID NOs: 1-35. Also provided are translational enhancer polynucleotides that comprise one or more of the specific TEEs exemplified herein or their variants, homologs or functional derivatives. Further provided are expression vectors comprising such TEEs or translational enhancer polynucleotides, as well as host cells and expression systems that harbor such vectors.

Description

[0001] References to Priority Documents [0002] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Serial No. 61 / 007,440, filed December 11, 2007. The filing date of the foregoing application is hereby regarded as the priority date of the present application, and the disclosures of the above provisional patent application are hereby incorporated by reference in their entirety. Background technique [0003] In eukaryotes, the translation process begins after the recruitment of the 40S ribosomal subunit, which can occur through the m7G capping structure, which is a 5' end of the mRNA, or through a capping-independent mechanism. Modified nucleotides discovered. The translation process that occurs through capping-independent mechanisms is defined as the internal initiation of the translation process that can be regulated by a number of different mechanisms. After recruitment by other mechanisms, the 40S ribosomal subunit moves to an ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/67
CPCC12N15/1051C12N15/67C12N15/85A61P43/00
Inventor V·P·莫罗G·M·伊德尔曼周伟
Owner THE SCRIPPS RES INST