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
2000-03-31
2003-09-09
Guzo, David (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, C435S252300, C435S252330, C536S023100, C536S024100, C536S024200
Reexamination Certificate
active
06617130
ABSTRACT:
BACKGROUND OF THE INVENTION
Recombinant DNA techniques have resulted in the production of heterologous polypeptides in bacterial hosts. However, the efficiency, regulation and polypeptide yield in such systems can vary depending on factors including the nature of the host bacteria, the type of heterologous polypeptide and the DNA fragments operably linked to the polypeptide coding sequence that control its expression.
One expression control element is the promoter, alternately referred to as a promoter region, which is a segment of DNA containing the site where RNA polymerase specifically binds and initiates RNA synthesis, or transcription, of the polypeptide coding sequence. The site on the promoter generally associated with RNA polymerase binding is referred to as the −35 consensus sequence and the promoter site generally associated with initiation of RNA synthesis by separation of the double stranded DNA into single stranded DNA for transcription is referred to as the −10 consensus sequence. Various promoters have been studied and used to express genes in bacterial hosts, including promoters that are not native to the bacterial host. For example, the trp and lac promoters have been isolated from
E. coli
and inserted into expression cassettes used to transform non-
E. coli
bacteria and express heterologous proteins. In order for transcription to take place, however, these non-native promoters must be recognized by the RNA polymerase of the host bacteria.
Not only must the expression system be recognized by the host cell, but the timing of the expression may need to be regulated to ensure that the heterologous protein production occurs at a suitable time, e.g., after sufficient growth and replication of host cells, at a suitable rate and for a sufficient period of time. For example, in a large scale fermentation, it may be desirable to control the production level of heterologous protein to prevent lethal overproduction in the host cell or interference with other cell functions, as well as to ensure optimal culture conditions and polypeptide recovery.
One naturally-occurring way for some bacteria to further control expression is by use of an operator element to regulate transcription. The operator is a segment of DNA operably connected to the promoter and acts as a binding site for a repressor protein or an activator protein. When a repressor protein binds to an operator, transcription from a promoter is shut off. Conversely, binding of an activator protein to an operator turns on transcription from a promoter. For both repressed and activated systems, transcription can be turned on by a process termed induction, in which the repressor protein is inactivated or in which the activator protein is activated. The net result of both types of induction is that RNA polymerase is able to bind to the promoter and initiate transcription. An expression control system involving a promoter/operator combination should be readily turned on and off and not be “leaky”, that is, producing a significant level of polypeptide either when the repressor protein is bound to an operator or when the activator protein is not bound to an operator.
Synthetic expression control systems have also been constructed that combine control elements from different sources. For example, Henner et al., U.S. Pat. No. 4,912,046, reports two hybrid promoter/operator constructs: (i) the pac-1 hybrid promoter/operator having its promoter RNA polymerase recognition site from the penicillinase promoter and an operator region from the lac promoter/operator and (ii) the spac-1 hybrid promoter/operator having its promoter RNA polymerase recognition site from a SPO-1 phage promoter and an operator from the lac promoter/operator. Henner et al. reports that the pac-1 hybrid promoter/operator was used to express
B. licheniformis
penicillinase in
Bacillus subtilis
and that the spac-1 hybrid promoter/operator was used to express leukocyte interferon in
Bacillus subtilis
. Additional hybrid promoter/operator systems are described by DeBoer, U.S. Pat. No. 4,551,433, which describes hybrid constructs having portions taken from the lac, trp and rrn promoter/operators. In these constructs, the promoter element is actually a hybrid promoter having its −10 consensus sequence contributed from one source and its −35 consensus sequence contributed from a different source.
Nonetheless, there is a need in the art for greater availability of expression control regions to regulate the expression of heterologous polypeptides in bacterial hosts, preferably exhibiting good expression control with high polypeptide yield.
SUMMARY OF THE INVENTION
This invention relates to materials and methods for expressing a heterologous polypeptide coding sequence in a bacterial host. The heterologous polypeptide is expressed under the control of a DNA construct containing a heterologous promoter region which is derived from a cyanophage or cyanobacteria promoter and which is operably linked to the coding sequence for the polypeptide. In one embodiment of the invention, such a promoter region is operably linked to an operator region that is derived from an operator native to the host cell. In another embodiment, the promoter region is operably linked to an operator region which is located upstream of the promoter region. In a specific embodiment, the invention utilizes a DNA construct for regulating expression of a heterologous polypeptide coding sequence in a transformed bacterial host cell, said DNA construct comprising (a) a promoter region derived from a cyanophage promoter, wherein the promoter region contains a −10 consensus sequence and a −35 consensus sequence, operably linked to (b) an operator region which is derived from an operator native to the host cell and which is located upstream of the promoter region. Each of these DNA constructs can be integrated into an expression vector which can be used to transform a bacterial host and enable production of the heterologous polypeptide.
The invention also encompasses methods of producing a heterologous polypeptide in a transformed bacterial host, comprising (a) stably transforming a bacterial host with a vector containing a heterologous polypeptide coding sequence operably linked to any of the above-mentioned DNA constructs for regulating expression of the coding sequence and (b) culturing the transformed bacteria under conditions that induce expression of the coding sequence.
The invention is also directed to expression vectors, transformed bacteria, and bacterial cultures containing such DNA constructs.
REFERENCES:
patent: 4551433 (1985-11-01), DeBoer
patent: 4704362 (1987-11-01), Itakura et al.
patent: 4861868 (1989-08-01), Krivi
patent: 4912046 (1990-03-01), Henner et al.
patent: 5162216 (1992-11-01), Scandella et al.
patent: 5221619 (1993-06-01), Itakura et al.
patent: 5516693 (1996-05-01), Vaeck et al.
patent: 5518897 (1996-05-01), Stevens, Jr. et al.
patent: 5583013 (1996-12-01), Itakura et al.
patent: 6194168 (2001-02-01), Gentz et al.
patent: 0067540 (1982-12-01), None
patent: 0108045 (1984-05-01), None
patent: 0971034 (2000-01-01), None
patent: WO 98/22590 (1998-05-01), None
No additional references are cited by the Examiner.*
Ma et al., Analysis of the Promoter and Regulatory Sequences of an Oxygen-Regulated bch Operon inRhodobacter capsulatusby Site-Directed Mutagenesis,Journal of Bacteriology, 175(7): 2037-45 (1993).
Schneider and Haselkorn, Characterization of Two Early Promoters of Cyanophage N-1;Virology167: 150-155 (1988).
Bogosian Gregg
O'Neil Julia P.
Terlesky Katherine C.
Beck George R.
Guzo David
Howrey Simon Arnold & White , LLP
Lambertson David A.
Monsanto Technology LLC
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