Cloning and expression of autogenes encoding RNA poly,erases of T7-like bacteriophages
First Claim
1. An autogene, comprising a gene encoding a T7-like bacteriophage RNA polymerase under the control of an autogene promoter recognized by said polymerase.
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Accused Products
Abstract
This invention relates to the cloning and expression of autogenes encoding RNA polymerases of T7 and T7-like bacteriophages, in which the RNA polymerase gene is transcribed from a promoter which is recognized by the encoded RNA polymerase. Cloning of T7 autogenes was achieved by reducing the activity of the RNA polymerase sufficiently to permit host cell growth. T7 RNA polymerase activity was controlled by combining two independent methods: lac-repression of the recombinant lac operator-T7 promoter in the autogene and inhibition of the polymerase by T7 lysozyme. Expression systems for producing the RNA polymerases of T7 and other T7-like bacteriophages, and expression systems for producing selected gene products are described, as well as other related materials and methods.
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Citations
32 Claims
- 1. An autogene, comprising a gene encoding a T7-like bacteriophage RNA polymerase under the control of an autogene promoter recognized by said polymerase.
- 2. An autogene, comprising a gene encoding bacteriophage T7 RNA polymerase under the control of an autogene promoter recognized by said polymerase.
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11. A construct for expressing the RNA polymerase of a T7-like bacteriophage, comprising:
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a) an autogene, which comprises a gene encoding a T7-like bacteriophage RNA polymerase under the control of an autogene promoter recognized by said polymerase; and b) a vector appropriate for maintenance of the construct in a host cell. - View Dependent Claims (12, 13, 14, 15, 16, 17, 18)
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19. An expression system for producing T7 RNA polymerase in host cells, comprising:
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a) an autogene construct, comprising; 1) an autogene, which comprises a gene encoding T7 RNA polymerase under the control of an autogene promoter comprising; a) a promoter recognized by said polymerase; and b) the lac operator, wherein the lac operator is centered at a position from +12 to +21 relative to the transcriptional start site; and 2) the lacI gene under the control of a second promoter; and b) a lysozyme construct, comprising a T7 lysozyme gene under the control of a third promoter. - View Dependent Claims (20, 21, 22, 23, 24)
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25. A method for producing an autogene, comprising:
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a) providing a first DNA fragment that contains the complete coding sequence for the RNA polymerase of a T7-like bacteriophage but no promoter that is recognized by said RNA polymerase; b) providing a second DNA fragment that contains a promoter that is recognized by said bacteriophage RNA polymerase; and c) joining said first and second DNA fragments so as to place the coding sequence for said bacteriophage RNA polymerase under control of said promoter that is recognized by said bacteriophage RNA polymerase, producing an autogene. - View Dependent Claims (26, 27, 28, 29, 30, 31, 32)
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Specification