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
1999-10-29
2003-02-18
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S004000, C435S015000, C435S023000, C435S034000, C435S069100, C435S252300, C435S320100, C536S023200, C536S023400
Reexamination Certificate
active
06521425
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to the screening for and use of agents which either inhibit or activate protein splicing of inteins (IVPS). Specifically, disclosed herein is the development of 2 specific reporter systems for Gyrase A and DnaB inteins. Agents screened for in accordance with the present invention can be used to control protein splicing for any purpose, in vivo or in vitro, including antimicrobial activity of organisms containing inteins in essential genes. More specifically, the present invention relates to the use of inteins expressed in modified or unmodified native protein splicing precursors or homologous extein systems to screen for mutations that modulate splicing or agents that inhibit or activate splicing. The present invention improves on current reporter systems used to screen for agents that can control splicing by using a modified or unmodified native precursor or precursor homolog in order to take advantage of the more native intein active site formed by natural precursors or inteins in homologous exteins, since agents that are derived from non-native precursors may not have the identical selected activity on native precursors.
Production of mature proteins involves the flow of information from DNA to RNA to protein. Precise excision of DNA and RNA elements which interrupt that information has been previously described (M. Belfort,
Annu. Rev. Genet.
24:363 (1990); T. R. Cech,
Annu. Rev. Biochem.
59:543 (1990); Hunter et al.,
Genes Dev.
3:2101 (1989)). More recently, evidence for the precise excision of intervening protein sequences has also been described for the TFPI allele from
Saccharomyces cerevisiae
(Hirata et al.,
J. Biol. Chem.
265:6726 (1990); Kane et al.,
Science
250:651 (1990)) and the recA gene from
Mycobacterium tuberculosis
(Davis et al.,
J. Bact.
173:5653 (1991); Davis et al.,
Cell
71:1 (1992)). Each of these genes contains internal in-frame peptide segments which must be removed to produce the mature protein. Expression of Tfp1 and RecA each results in two peptides: one representing the intervening protein sequence (IVPS) and the other the ligated product of the external protein sequences (EPS). In 1994, the terms “intein” and “extein” were adopted in place of IVPS and EPS, respectively (Perler, et al., Nucleic Acids Res. 22:1125-1127 (1994)). This post-translational processing event has been termed “protein splicing”. Similarly, the “Vent”® DNA polymerase gene from the hyperthermophilic archaeon
Thermococcus litoralis
contains two in-frame IVPS (Perler, et al., PNAS 89:5577 (1992)) and the DNA polymerase gene from the hyperthermophilic archaeon Pyrococcus species GB-D contains one intein (Xu, M., et al., Cell 75, 1371-1377 (1993)).
Over 80 inteins have been identified in bacteria, archaea and eucarya (Perlèr, F. B., et al. Nucleic Acids Res 25, 1087-93 (1997), Dalgaard, J. Z., et al., J Comput Biol 4, 193-214 (1997), Pietrokovski, S., Protein Sci. 7, 64-71 (1998) and Perler, F. B. Nucleic Acids Res. 27, 346-47 (1999). Four inteins have been found in
Mycobacterium leprae
(Davis, E. O., et al., EMBO J. 13, 699-703 (1994) and Smith, D. R., and et al. Genome Res 7, 802-19 (1997)) and three inteins in
Mycobacterium tuberculosis
(Cole, S. T., et al. . Nature 393, 537-44 (1998)). One intein has been found in
Candida tropicalis
(Gu, et al., J. Biol. Chem., 268(10):7372-7381 (1993)).
Controllable IVPS (CIVPS) and methods for using the same to modify, produce and purify target proteins has been described (Comb et al., U.S. Pat. No. 5,496,714, issued Mar. 5, 1996; Comb et al., U.S. Pat. No. 5,834,247, issued Nov. 10, 1998). Methods for using inteins to screen for peptides (or derivative, analogic or mimetic thereof) or any agent that can enter cells to block or activate splicing of a natural or experimental reporter protein have also been described (U.S. Pat. No. 5,834,247, supra. at Example 17). These methods specifically describe the screening of peptides using mycobacterial inteins as targets. The preparation of an in vivo peptide library utilizing chicken &agr;-spectrin is also described.
While a general method of screening for antimicrobial agents using the
M. tuberculosis
RecA intein in a thymidylate synthetase (TS) reporter system has been described (Belfort, U.S. Pat. No. 5,795,731, issued Aug. 18, 1998), this system suffers from several limitations. Importantly, several studies of protein splicing in foreign contexts (such as the Belfort system) indicate that intein splicing is more efficient in the native extein than in foreign exteins (Xu, EMBO J. 13:5517-5522 (1994), Xu, EMBO J. 15:5146-5153 (1996), Telenti, J. Bacteriol. 179:6379-6382 (1997), Chong J. Biol. Chem, 273:10567-10577 (1998), Liu, FEBS Lett. 408:311-314 (1997), Wu, Biochim. Biophys. Acta 1387:422-432 (1998B), Nogami Genetics, 147:73-85 (1997), Kawasaki J. Biol. Chem., 272:15668-15674 (1997), Derbyshire, Proc. Natl. Acad. Sci USA, 94:11466-11471 (1997), Southworth, BioTechniques 27:110-121 (1999), FIG.
7
)). For example, the use of foreign exteins yields temperature-dependent splicing of the Psp-GBD Pol, Mxe GyrA and Synechocystis DnaB inteins (Xu, EMBO J. 13:5517-5522 (1994), Xu, EMBO J. 15:5146-5153 (1996), Telenti, J. Bacteriol. 179:6379-6382 (1997), Chong J. Biol. Chem, 273:10567-10577 (1998), Liu, FEBS Lett. 408:311-314 (1997), Wu, Biochim. Biophys. Acta 1387:422-432 (1998B), Nogami Genetics, 147:73-85 (1997), Kawasaki J. Biol. Chem., 272:15668-15674 (1997) and Southworth, BioTechniques, 27:110-121 (1999), and FIG.
7
).
While not wishing to be bound by theory, it is believed that such inefficient protein splicing in the foreign extein context occurs because the flanking extein is, in effect, the substrate of the intein. It is, therefore, likely that the intein may exhibit substrate specificity like all other enzymes. The substrate specificity of the intein limits acceptable extein sequences, hence the native extein sequence is the optimal substrate, whereas foreign extein sequences may not be acceptable substrates at all. For example, studies of the Sce VMA and Mxe GyrA inteins indicate that thiol induced N-terminal splice junction cleavage and splicing are, to varying extents, dependent on the single extein residue preceding the intein (Chong, J. Biochem. 273:10567-10577 (1998), Southworth, BioTechniques, 27:110-121 (1999)). Other extein residues have also been shown to affect splicing of the Sce VMA intein (Nogami Genetics, 147:73-85 (1997), Kawasaki J. Biol. Chem., 272:15668-15674 (1997)).
Additionally, exteins may affect the packing at the intein active site, or global folding of the intein and/or precursor, hence the use of a foreign extein may result in improper folding of the intein or precursor and inefficient or no splicing. Moreover, expression of an extein gene that naturally contains an intein in a foreign host, for example
E. coli
or yeast, may not be efficient (Perler et al. Proc. Natl. Acad. Sci. USA 89:5577-5581 (1992) and Hodges, et al., Nucleic Acid Res. 20:6153-6157 (1992)), whereas expression of the homologous endogenous extein is likely to be more efficient. For example, the
Mycobacterium leprae
RecA intein fails to splice in
E. coli
, while it splices in
M. leprae
(Davis, et al., EMBO J., 13:699-703 (1994)). It is possible that the
M. leprae
RecA intein would splice in
E. coli
RecA, although that has yet to be tested. In another example, the Synechocystis sp. strain PCC6803 DnaB gene, containing an intein, was unclonable in
E. coli
( Wu, et al., Proc. Natl. Acad. Sci. USA 95:9226-9231 (1998)). The
M. leprae
GyrA precursor did not splice efficiently in
E. coli
and was mostly insoluble, while the homologous Mxe GyrA intein spliced efficiently in
E. coli
GyrA.
Additionally, the use of homologous exteins would eliminate, in many instances, the need to introduce silent mutations in the reporter gene in order to insert the desired intein (see Belfort, supra., Comb, supra, Example 17).
Homologous exteins may have naturally-occuring, conserved restriction enzyme sites that would
Adam Eric E.
Perler Francine B.
Achutamurthy Ponnathapu
Moore William W.
New England Biolabs Inc.
Strimpel Harriet M.
Williams Gregory D.
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