Translation enhancer-element dependent vector systems
First Claim
1. A nucleic acid vector comprising a first construct comprising two or more first transcriptional elements, a first cistron encoding a transcription factor, and one or more first translational enhancer elements (TEEs), wherein the transcription factor amplifies the transcription of at least one cistron of the first construct.
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Abstract
A translation enhancer-driven positive feedback vector system is disclosed which is designed to facilitate identification of a Translational Enhancer Element (TEE) and to provide a means for overexpression of gene products. The system exploits both transcriptional and translational approaches to control the expression levels of genes and/or gene products. Methods are also disclosed for screening libraries of random nucleotide sequences to identify translational elements and for overproduction of proteins, which have uses in both research and industrial environments.
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Citations
55 Claims
- 1. A nucleic acid vector comprising a first construct comprising two or more first transcriptional elements, a first cistron encoding a transcription factor, and one or more first translational enhancer elements (TEEs), wherein the transcription factor amplifies the transcription of at least one cistron of the first construct.
- 20. A nucleic acid vector comprising a first construct comprising a first transcriptional element, a first cistron encoding a first gene product, and a first translational enhancer element (TEE), wherein the TEE is resistant to an activity of a second gene product which blocks host protein synthesis.
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26. A method of identifying a translational enhancer element (TEE) comprising:
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a) inserting nucleotides from a library of nucleotides into a vector comprising a first construct comprising two or more first transcriptional elements, and a first cistron encoding a transcription factor;
b) transfecting a cell with the vector of step (a);
and c) determining the level of gene product translation from one or more second constructs in the cell of step (b), wherein determining an enhanced level of translation of the gene product in the presence of the inserted nucleotides is indicative of the presence of at least one TEE. - View Dependent Claims (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 45, 46, 47)
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38. A method of overexpressing a gene comprising:
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a) transfecting a cell with a vector comprising a first construct comprising two or more first transcriptional elements, a first cistron encoding a transcription factor, and one or more first translation enhancer elements (TEEs); and
b) expressing, in the cell of step (a), a gene product from one or more second constructs comprising a second TEE, wherein the resulting level of gene product expressed from the second construct is enhanced in the presence of the first and second TEEs. - View Dependent Claims (39, 40, 41, 42, 43, 44)
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48. A method of overexpressing a gene comprising:
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a) transfecting a cell with a vector comprising a first construct comprising at least one first transcriptional element, a first cistron encoding a first gene product, and at least one translational enhancer element (TEE); and
b) expressing, in the cell of step (a), a gene from one or more second constructs encoding a protein which blocks host protein synthesis with one or more TEEs, wherein the resulting level of gene product expressed from the first construct is enhanced in the presence of the blocking protein. - View Dependent Claims (49, 50, 51, 52, 53, 54, 55)
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Specification