Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
2000-01-06
2004-03-02
Leffers, Jr., Gerald G. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S023000, C435S091400, C435S320100, C435S069700, C435S069100, C435S252330, C435S006120, C435S007200, C435S007370, C435S007400, C435S024000, C536S023100, C536S023400, C536S024100, C530S350000
Reexamination Certificate
active
06699702
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a prokaryotic cell system and cell based assays for: identification of protease inhibitors and protease modulators; ii) determination of the amino acid sequence of a cleavage site for a known protease; iii) identification and cloning of a protease whose cleavage site is known; and iv) rapid identification of a form of a protease that exhibits increased protease activity relative to a control protease.
BACKGROUND OF THE INVENTION
Proteases are enzymes that cleave peptide bonds, and hence by definition, alter proteins. These enzymes are broadly classified into four groups: a) serine proteases, b) cysteine proteases, c) aspartate proteases and d) metalloproteases (Nduwimana et al., Ann. Biol. Clin. 53:251-264 (1995). This classification is primarily based on the mechanism of action. Many of these proteases which cleave peptide bonds at specific sites have been implicated in a variety of human diseases (Melnick, J. L., in Virology 1985, B. N. Fields, Ed. Raven Press, New York, pp. 739-794; Ratner et al., Nature (1985) 313:277-283; Farmerie et al., Science (1987) 236:305-308; Imai, T., et al., J. Biochem. (1986) 100:425-432; Cox, D. W. et al., Am. Rev. Respir. Dis., (1988) 137:371-375, Albin, R. J. et al., Am. Rev. Respir. Dis., (1987) 135:1281-1285) These diseases include hypertension, onset of emphysema, several neurological disorders, onset of respiratory diseases and several autoimmune diseases. Proteases have also been shown to play a very important role in the physiology of several pathogenic microorganisms and the maturation of viruses (Toyoda et al., Cell (1986) 45:761., Hanecak et al., Cell (1984) 37:1063; Kohl, N. E., et. al., Proc. Nat. Acad. Sci. (USA) (1988) 85:4686-4690)) Hence, proteases are also implicated in the establishment of infectious diseases.
In all of these cases, it is believed that a specific inhibitor or a modulator of a specific protease may serve as a therapeutic agent that can be used to either prevent a disease or help control and mitigate the adverse effects of a disease. This has led to an enormous effort to discover these specific protease inhibitory agents or agents that help modulate protease activity. Conventional methods in the past have used kinetic enzyme assays that monitor the cleavage of either a radiolabeled substrate or a chromogenic substrate (Billich, S., et al., J. Biol. Chem., (1988) 263:17905-17908, Moore, M. L., et. al. Biochem. Biophys. Res. Commun. (1989) 159:420-425; Blumenstein J. J., et al., Biochem. Biophys. Res. Commun. (1989) 163:980-987;). Other methods have also been used to monitor protease activity, however, all of these techniques are time consuming, cumbersome and labor intensive (Dreyer G. B. et al. Proc. Nat. Acad Sci. (USA) (1989) 86: 9752-9756; Krausslich, H. et al. Proc. Natl. Acad. Sci. (USA) (1989) 86: 807-811; Drake, P. L., et al., Biochem. Biophys. Res. Commun. (1988) 156:297-303). None of these methods could be used in a high throughput format to evaluate millions of compounds that can be generated using automated combinatorial chemistry. More recently, whole cell based enzyme assays have been developed by engineering a cleavage site within the structural portion of a particular enzyme or an efflux pump (Baum, E. Z. et al., (1990) Proc. Natl. Acad. Sci. (USA) (1990) 87:10023-10027; Block, T. M. et al., Antimicro. Ag. Chemo. (1990) 34:2337-2341). These assays are more compatible with high throughput screening, however they are not sensitive and versatile. It has long been realized that a robust, versatile and sensitive assay that can be used in a homogenous format and is compatible with high-throughput screening would be highly beneficial in the effort to identify inhibitors and modulators of protease activity.
In many of the disease cases mentioned above it has long been established that a specific protease activity is implicated (Black, R. A., et al., Nature (1997) 385:729733). However, the protease gene has not been cloned. A rapid and reliable method has been needed to screen a genomic or cDNA library to clone the protease. Conventional molecular biological techniques have been used in the past with some degree of success. Also, in many cases the specific cleavage sequence of a particular protease is not known. At the present time a rapid and reliable method is not available to screen a library of protease cleavage sites (WO 96/21009).
Finally, in the field of protein structural studies there has always been a need for generating large quantities of the protein being studied. Heterologous gene expression systems utilizing the bacteria
Escherichia coli
(
E. coli
), or the yeast
Picchia pastoris
or the insect virus Bacculovirus have been widely used. Unfortunately, in very many cases the heterologous protein is found to be sequestered in insoluble aggregates called inclusion bodies or is found in some other precipitated or aggregated inactive form. In many cases, a slight change in the amino acid sequence of the protein results in the production of a soluble and hence active protein. The change is subtle enough so that the integral structure or the activity of the protein remains unaffected. However, identification of these subtle solubilizing changes is often very cumbersome and labor intensive. A rapid method has long been sought to screen a library of mutant proteases and identify the ones that are expressed in a more soluble and hence active form.
SUMMARY OF THE INVENTION
The present invention relates to a prokaryotic cell system for monitoring protease activity that can be used to i) identify protease inhibitors and protease modulators, ii) determine the sequence of a protease cleavage site for a known protease, iii) identify and clone the gene for a protease whose cleavage site is known, and iv) rapidly identify a form of a protease that exhibits protease activity when the wild type protease exhibited little or no activity in a prokaryotic system.
One aspect of the invention is a prokaryotic cell system for monitoring protease activity comprising a prokaryotic cell comprising:
a) a gene coding for a protease;
b) a modified DNA-binding repressor gene wherein said modification is one or more cognate cleavage sites of the protease engineered into one or more exposed and permissive loops of the repressor polypeptide encoded by said repressor gene; and
c) a reporter cartridge wherein the expression of the reporter gene in said reporter cartridge is regulated by a promoter whose transcription can be negatively regulated by the wild type or the modified DNA-binding repressor protein.
Using this system, the activity of a protease can be determined by monitoring the level of expression of the reporter gene; i.e., the reporter gene activity. The modified or the wild-type repressor negatively regulates the expression of the reporter gene. If the protease is expressed in the same cell and is active, it cleaves the modified repressor at its cognate cleavage site, which has been engineered into the repressor. The cleaved repressor is not able to bind to its cognate DNA sequence and hence does not regulate the expression of the reporter gene. Thus by monitoring the activity of the reporter gene, we can monitor the activity of the protease.
Another aspect of the invention is an assay for identifying protease inhibitors and protease modulators. This can be achieved by using the above mentioned prokaryotic cell system capable of expressing a protease, a modified DNA-binding repressor containing the cognate cleavage site, and the appropriate reporter cartridge. The cell can be grown in the presence of a potential inhibitor or potential modulator. Upon incubation and allowing for the expression of the protease, transcription of the reporter gene can be quantified. The level of expression can be used to correlate to the efficacy of the potential protease inhibitor or potential protease modulator.
Another aspect of the invention is an assay to determine a cleavage site of a known protease whose cleavage site is not known. This can be achieved, for instance, in the foll
Lach David
Patel Pramathesh
Bristol-Myers Squibb Co.
Leffers, Jr. Gerald G.
Sher Audrey F.
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