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
1999-04-01
2001-04-17
Ketter, James (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...
C435S320100, C435S325000, C435S252330, C435S410000, C435S476000, C435S194000, C435S483000, C435S485000
Reexamination Certificate
active
06218145
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of expression of desired, protein-encoding genes in a host cell. More particularly, it concerns expression of desired heterologous, e.g., mammalian, protein-encoding genes in a host cell wherein the heterologous gene is operably linked to a heterologous promoter recognized by a heterologous RNA polymerase, wherein the gene encoding the heterologous RNA polymerase is expressed in the host cell under the control of a host cell promoter. It also concerns ectopic expression in a host cell of desired native genes operably linked to a heterologous promoter recognized by a heterologous RNA polymerase, wherein the gene encoding the heterologous RNA polymerase is expressed in the host cell under the control of a host cell promoter.
2. Description of Related Art
Recombinant DNA technology allows insertion of a desired heterologous protein-encoding gene, which may also be referred to herein as a “target gene,” into a host cell and subsequent expression of the gene product of the protein-encoding gene.
U.S. Pat. Nos. 4,704,362; 5,221,619; and 5,583,013 (Itakura et al.) disclose a method for expressing, in a bacterial host, proteins that are “heterologous” with respect to the host, i.e., not ordinarily produced by the host, using expression control systems that are “homologous” to the bacterial host, i.e., systems that are ordinarily produced by the bacterial host in its untransformed state. There are various reasons to provide new methods for expressing heterologous or, less commonly, homologous proteins in microbial hosts using a promoter system that is heterologous with respect to the host. However, some heterologous promoter systems that are advantageous for this purpose may not be recognized by the host's RNA polymerases and hence, without more, are not able to drive such expression. This invention provides a method for making such heterologous promoter systems useful to drive expression of such proteins in microbial and other hosts, by providing an RNA polymerase capable of recognizing the heterologous promoter.
A disadvantage of the homologous regulon system is the promoter recognized by the host RNA polymerase would be expected to drive expression of genes native to the host cell in addition to the protein-encoding gene. The presence of host gene products may decrease the purity of the target gene product and may require time-consuming and expensive steps for the removal of the host gene products. Therefore, it is desirable to have a system for expressing a protein-encoding gene in a host cell wherein the protein-encoding gene is under control of a promoter heterologous to the host cell, and as a result the protein-encoding gene can be expressed to yield greater purity of gene product. Such a system may be termed a “heterologous regulon.” In a heterologous regulon system, it is necessary to provide a heterologous RNA polymerase that recognizes the promoter heterologous to the host cell and drives expression of the protein-encoding gene.
SUMMARY OF THE INVENTION
The present invention provides a vector system for expressing a protein-encoding gene in a host cell. The vector system comprises a plant organellar genomic promoter operably linked to the protein-encoding gene, and a second promoter operably linked to a gene encoding an RNA polymerase capable of recognizing said plant organellar genomic promoter. Preferably, the RNA polymerase of the vector system is heterologous to the host cell, i.e. not normally found in the host cell in the untransformed state. The RNA polymerase of the vector system may also be referred to as the “heterologous RNA polymerase.” The plant organellar genomic promoter is not recognized by RNA polymerases of the host cell, but is instead recognized by the RNA polymerase of the vector system. In a preferred embodiment, the RNA polymerase of the vector system is a single-subunit RNA polymerase capable of recognizing promoters found in the genomes of plastids or plant mitochondria. In a further preferred embodiment, the heterologous RNA polymerase is under the control of an inducible promoter recognized by a host cell RNA polymerase.
In accordance with one aspect of the present invention, double-stranded DNAs (dsDNAs) comprising the plant organellar genomic promoter operably linked to the protein-encoding gene and the second promoter operably linked to the heterologous RNA polymerase gene are both present on single vector. In accordance with another aspect of the present invention, the dsDNAs comprising he plant organellar genomic promoter operably linked to the protein-encoding gene and the host cell promoter operably linked to the heterologous RNA polymerase gene are each present on separate vectors. In accordance with yet another aspect of the present invention, one of the dsDNAs comprising the plant organellar genomic promoter operably linked to the protein-encoding gene and the host cell promoter operably linked to the heterologous RNA polymerase gene is integrated into a host cell chromosome and the other dsDNA is present on a vector. In accordance with still another aspect of the present invention, both dsDNAs comprising the plant organellar genomic promoter operably linked to the protein-encoding gene and the host cell promoter operably linked to the heterologous RNA polymerase gene are integrated into a host cell chromosome.
The present invention also provides for recombinant host cells comprising dsDNAs in accordance with the above-mentioned elements. In a preferred embodiment, the recombinant host cell is a microbe. In a further preferred embodiment, the microbe in question is
Escherichia coli
strain K-12 W3110 or a derivative thereof. In accordance with still another aspect of the present invention, a method is provided for expressing a protein-encoding gene in a host cell, using the above-mentioned elements.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The expression in a host cell of a protein-encoding gene which exists in dsDNA form involves transcription of messenger RNA (mRNA) from one strand of the DNA by an RNA polymerase enzyme. Subsequently the mRNA is processed, involving recognition of a region of the 3′ UTR and addition of a tail of polyadenylate nucleotides to the 3′ end of the mRNA by polyadenylation enzymes. Finally, the mRNA encounters ribosomes which associate to a region of the 5′ UTR of the mRNA and then translocate in a 3′-ward direction along the mRNA. During translocation, amino acids are added to one another in sequence to form the polypeptide product of the protein-encoding gene.
Transcription of DNA into mRNA is regulated by a region of DNA usually referred to as the promoter. The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA, and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding complementary strand of RNA. RNA polymerases from different species typically recognize different sequences of bases as promoter regions. In order to express a protein-encoding gene in a host cell, either the promoter driving transcription of the protein-encoding gene must be recognized by a host RNA polymerase, or an RNA polymerase which recognizes the promoter driving transcription of the protein-encoding gene must be provided to the host cell.
Most work in the field has focused on expression systems in which the promoter driving transcription of the protein-encoding gene is replaced with one recognized by host RNA polymerase. An example of such a system is the homologous regulon disclosed by U.S. Pat. Nos. 4,704,362; 5,221,619; and 5,583,013 (Itakura et al.). The approach of the present invention is to import into the host cell both a protein-encoding gene under control of a heterologous promoter and a gene or genes encoding a heterologous RNA polymerase that recognizes the heterologous promoter. The gene or genes for the heterologous RNA polymerase must be under control of a promoter recognized by a h
Bogosian Gregg
O'Neil Julia P.
Staub Jeffrey M.
Arnold & White LLP
Beck, Esq. George R.
Ketter James
Monsanto Company
Simon Howrey
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