Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
2000-12-21
2004-10-12
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S455000, C435S252300, C435S252340, C435S320100, C536S023100, C536S023200, C536S024100, C536S024200
Reexamination Certificate
active
06803224
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the design of transcriptional cascade circuits to amplify gene expression. It also relates to the use of these systems for the overproduction of polypeptides such as therapeutic proteins, enzymes, hormones, growth factors, and apoliproteins in vitro, and in cells, i.e., cell cultures. This has great industrial utility, e.g., in the biotechnology and pharmaceutical industries.
BACKGROUND OF THE INVENTION
Overexpression of cloned genes is very convenient for production of either recombinant polypeptides or specific cell metabolites for basic research, and the pharmaceutical and biotechnology industries generally. Production of large amounts of cloned genes has traditionally been achieved by combining gene amplification with strong promoters regulated by repressors. However, these conventional strategies typically have the disadvantages that: 1) maintenance of plasmid expression vectors typically requires selection with antibiotics, giving rise to metabolic burden and additional costs for large scale industrial production (Nilson and Skogman 1986); 2) low level expression is indicated when dealing with toxic proteins, and in avoiding accumulation of mutations in the recombinant protein products themselves (Mertens et al., 1995; Vilette et al. 1995), which is difficult to achieve when the cloned gene is in multicopy due to the multiple copy number of traditional plasmid vectors and the high basal (“leaky”) level of expression of most traditionally used promoters, e.g., tac or trc; 3) traditional inducers, e.g., IPTG in lac expression systems, are expensive and have a certain degree of toxicity (Figge et al. 1988); 4) high expression of recombinant proteins has been shown to reduce host cell growth rate and, concomitantly, overall protein synthesis (Bentley et al., 1990; Dong et al. 1995), presumably due to increased metabolic burden on the host; and 5) a considerable number of existing expression systems only replicate in
E. coli
, which may limit expression of certain proteins, e.g., those desired to be secreted.
An alternative expression system that fulfills some of the above requirements uses miniTn5 transposon vectors (de Lorenzo and Timmis 1994) to insert heterologous genes into the bacterial chromosome, thereby allowing high stability of expression (Cebolla et al. 1993; Suarez et al. 1997). Suarez in particular describes the stable production of pertusic toxin in
Bortedetella bronchiseptica
by miniTn5-mediated chromosomal insertion and expression using a salicylate regulatory system. Salicylate is a benzoate inducer 1000-fold less expensive than IPTG (SIGMA catalog 1998). The system is based on the nahR regulatory gene, which encodes a positive regulator activated by salicylate and its target promoter Psal (de Lorenzo et al. 1993). However, expression levels obtained are relatively poor (0.1% of total proteins). This low level is likely because the genes are in monocopy in the chromosome.
If yield could be improved while maintaining the advantages of low basal levels, stability, broad host range, and low cost, the nahR/Psal regulatory system would have great industrial utility.
SUMMARY OF THE INVENTION
We have designed a cascade system that allows 10 to 20 fold greater expression over the standard nahR/Psal system while substantially retaining one or more of that system's innate advantages. To achieve this, another regulatory element, xylS2 and its target promoter Pm, is coupled to Psal expression in a cascade circuit. The xylS2 regulatory gene responds to the common inducer and has more gene expression capacity than standard nahR/Psal. Synergistic activation of the Pm promoter by the XylS2 transcriptional activator can be achieved by simultaneously increasing the intracellular concentration and specific activity of activator/regulator in the presence of a common benzoate derivative inducer, e.g., salicylate.
Accordingly, in a first aspect the invention features a cascade genetic circuit comprising one or more nucleic acid constructs encoding a plurality of transcriptional regulators, said encoded regulators arranged in a hierarchical order such that expression of an upstream regulator from said plurality stimulates expression of a downstream regulator from said plurality; and a final target promoter, said final target promoter responsive in a dose-dependent fashion to a terminal downstream regulator of said plurality of regulators.
In certain preferred embodiments, it may be useful to introduce the final target promoter alone, e.g., via PCR, into a host genome at designated position to determine effect on expression of the downstream sequence. To do this, it may first be desirable to disable or knock out the native promoter, gene, or nucleic acid sequence. In other preferred embodiments, as described below, heterologous genes and sequences are preferred for use with the cascade circuit and, accordingly, may be introduced.
In one especially preferred embodiment, the cascade genetic circuit further comprises a multiple cloning site downstream of the final target promoter.
In another preferred embodiment, the cascade genetic circuit, or at least a portion thereof, is present as a chromosomal integration in a host cell. In a different, not necessarily mutually exclusive embodiment, at least one of said one or more nucleic acid constructs is present as an autoreplicative plasmid.
In a further embodiment, the cascade genetic circuit, or at least a portion thereof, is responsive to an inducer, preferably an inducer that is capable of inducing the expression of more than one regulator in the cascade. In preferred embodiments, the inducer is a benzoate derivative, preferably, although not necessarily, salicylate.
In another aspect, the invention features a cell, tissue, or organism comprising the cascade genetic circuit of any of the preceding claims. Preferably, the cell is selected from the group consisting of procaryotic and eukaryotic cells. As concerns eukaryotic cells, mammalian, insect, yeast, and plant cells are preferred. As concerns procaryotic cells, gram-negative bacterial cells are preferred.
In yet another aspect, the inventon features methods of regulating the expression of a nucleic acid sequence, comprising establishing a cascade genetic circuit according to any of the cascade genetic circuit embodiments described above; placing said nucleic acid sequence under control of said final target promoter; and inducing said cascade genetic circuit to stimulate expression of said nucleic acid sequence.
Preferably the nucleic acid sequence encodes a polypeptide selected from the group consisting of enzymes, hormones, growth factors, apolipoproteins, therapeutic proteins, diagnostic proteins, and portions or derivatives thereof. In other preferred embodiments, the nucleic acid sequence encodes an anti-sense molecule, ribozyme, rRNA, tRNA, snRNA, or simply a diagnostic RNA molecule. In certain preferred embodiments, the nucleic acid encodes a reporter gene product useful in diagnostics.
DETAILED DESCRIPTION OF THE INVENTION
By using regulatory genes of the control circuits for the expression of catabolic operons, a cascade expression system for amplifying gene expression was constructed. The system is based on the activation characteristics of the Pm promoter by the XylS2 mutant transcriptional activator. Strength of Pm activation depends on both the amount of XylS2 protein and its specific activity, which is enhanced by the presence of salicylate and other benzoate derivatives. To couple the increase of XylS2 intrinsic activity and XylS2 intracellular concentration, the expression of xylS2 is under the control of the Psal promoter and the NahR transcriptional activator, that is also activated in response to common inducers. The synergistic action of both transcriptional regulators lead to 10 to 20-fold amplification of the gene expression capacity with regard to each individual expression system.
One embodiment of the system comprises a cassette having the regulatory genes nahR/Psal::xylS2 flanked by transposabl
de Lorenzo Prieto Victor
Martin Carolina Sousa
Ramirez Angel Cebolla
Bio Technology Law Group
Chambers Daniel M.
Counsejo Supeslor de Investigaciones Cientificas
Guzo David
Kreusser Edward O.
LandOfFree
Regulated expression of cloned genes using a cascade genetic... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Regulated expression of cloned genes using a cascade genetic..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Regulated expression of cloned genes using a cascade genetic... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3306969