Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-11-30
2002-07-16
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S243000, C435S320100, C435S325000, C435S410000, C536S024100
Reexamination Certificate
active
06420138
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to highly efficient and highly repressible expression control sequences, expression vectors which contain these expression control sequences, microorganisms transformed with these expression vectors, and methods for their production by means of recombinant DNA technology. The present invention also relates to methods for the production of viral, prokaryotic, and eukaryotic proteins and fragments thereof using these highly repressible expression control sequences, expression vectors and transformed microorganisms.
BACKGROUND OF THE INVENTION
The level of production of a protein in a host cell is determined by three major factors: the number of copies of its structural gene within the cell, the efficiency with which the structural gene copies are transcribed and the efficiency with which the resulting messenger RNA (“mRNA”) is translated. The transcription and translation efficiencies are, in turn, dependent on nucleotide sequences which are normally situated ahead of the desired structural genes or the translated sequence. These nucleotide sequences (expression control sequences) define, inter alia, the location at which the RNA polymerase binds (the promoter sequence to initiate transcription; see also EMBO J. 5:2995-3000 (1986)) and at which the ribosomes bind and interact with the mRNA (the product of transcription) to initiate translation.
Not all expression control sequences have the same efficiency. It is therefore often advantageous to separate the specific coding sequence for a desired protein from its adjacent nucleotide sequences and to link it with other expression control sequences to achieve a higher expression rate. After this linkage has been accomplished, the newly combined DNA fragment can be inserted into a plasmid capable of achieving a high copy number or a derivative of a bacteriophage to increase the structural gene copies within the cell, thereby improving the yield of the desired protein.
The constitutive expression of overproduction of both toxic and normally nontoxic gene products is often harmful to the host cells, thereby, lowering the stability of a specific host cell-vector system. Therefore, an expression control sequence should, in addition to improving the transcription and translation efficiency of a cloned gene, be regulatable to permit the regulation of the expression during the growth of the microorganisms. Some regulatable expression control sequences can be switched off during the growth of the host cells and then can be switched on again at a desired point in time, to favor the expression of large amounts of the desired protein.
Various expression control sequences have been used to regulate the expression of DNA sequences and genes which code for desired proteins. See, e.g., Itakura et al.(1977) Science 198:1056-1063; Goeddel et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76:106-110; Emtage et al. (1980) Nature 283:171-174; Bernard et al. (1979) Sciences 205:602-607; Ammann et al. (1983) Gene 25:167-178; de Boer et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80:21-25; European Patent Application Publication Nos. 41767 and 186069.
SUMMARY OF THE INVENTION
In accordance with the invention, highly efficient and highly repressible expression control sequences can be produced by combining promoter sequences with operator/repressor sequences. The present invention provides for expression control sequences for the expression of polypeptides from a gene or polynucleotide of interest. The gene or polynucleotide may express a viral, prokaryotic, or eukaryotic polypeptide. These control sequences are produced by combining bacterial or phage promoter and operator/repressor systems.
More particularly, the present invention provides for DNA expression control sequences comprising at least a portion of one or more lac operator sequences and a phage promoter comprising a −30 region, having a sequence represented by the sequence 5′ TTGMYW 3′ (where M=A or C and Y=C or T and W=A or T), and a −12 region, having a sequence represented by the sequence 5′ TAWRMT (where R=A or G).
The present invention further provides for DNA expression control sequences comprising at least a portion of one or more lac operator sequences and a phage promoter comprising a −30 region, having a sequence represented by the sequence 5′ TTGAYA 3′ (where Y=C or T), and a −12 region, having a sequence represented by a sequence selected from the group consisting of: 5′ TAWRTT 3′ (where R=A or G), 5′ TAWGMT 3′ (where W=A or T and M=A or C), and 5′ TAARMT 3′.
The present invention further provides for DNA expression control sequences comprising at least a portion of one or more lac operator sequences and a phage promoter comprising a −30 region, having a sequence represented by the sequence 5′ TTGMTW 3′ (where M=A or C and W=A or T), and a −12 region, having a sequence represented by a sequence selected from the group consisting of: 5′ TAWRTT 3′ (where R=A or G), 5′ TAWGMT 3′, and 5′ TAARMT 3′.
The present invention further provides for DNA expression control sequences comprising at least a portion of one or more lac operator sequences and a phage promoter comprising a −30 region, having a sequence represented by the sequence 5′ TTGCTW 3′ (where Y=C or T and W=A or T), and a −12 region, having a sequence represented by a consensus sequence selected from the group consisting of: 5′ TAWRTT 3′ (where R=A or G), 5′ TAWGMT 3′ (where M=A or C), and 5′TAARMT 3′.
The present invention further provides for DNA expression control sequences comprising at least a portion of one or more lac operator sequences and a phage promoter comprising a −30 region, having the sequence 5′ TTGACT 3′, and a −12 region, having the sequence 5′ TAAGAT 3′.
The present invention further provides for expression vectors comprising the above expression control sequences, host cells transformed with such expression vectors, and methods for producing viral, prokaryotic, and eukaryotic polypeptides using the disclosed expression control sequences.
REFERENCES:
patent: 4689406 (1987-08-01), Banks et al.
patent: 4868111 (1989-09-01), Bujard et al.
patent: 5362646 (1994-11-01), Bujard et al.
patent: 5629205 (1997-05-01), Lagosky
patent: 6194168 (2001-02-01), Gentz et al.
McClure, “Mechanism and Control of Transcription Initiation in Prokaryotes,”Ann. Rev. Biochem.,54:171-204 (1985).
Hawley et al., “Compilation and analysis ofEscherichia colipromoter DNA sequences,”Nucleic Acids Research, 11(8):2237-2255 (1983).
Horii et al., “Regulation of SOS Functions: Purification ofE. coliLexA Protein and Determination of Its Specific Site Cleaved by the RecA Protein,”Cell, 27(2):515-522 (1981).
Siebenlist et al., “E. coliRNA Polymerase Interacts Homologously with Two Different Promoters,”Cell, 20:269-281 (1980).
Roberts et al., “A general method for maximizing the expression of a cloned gene,”PNAS(USA), 76(2):760-764 (1979).
International Search Report mailed on Feb. 3, 1999, in International application No. PCT/US98/20075.
Coleman Timothy A.
Gentz Reiner L.
Human Genome Sciences Inc.
Human Genome Sciences, Inc
McKelvey Terry
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