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Inositolphosphoryl ceramide (IPC) synthase genes from fungi

a technology of inositolphosphoryl ceramide and fungi, which is applied in the direction of transferases, enzymology, microorganisms, etc., can solve the problems of not possessing clinically significant activity and no clinically useful compounds at this step, and achieves high stringency, high stringency, and disfavorable high stringency conditions.

Inactive Publication Date: 2002-05-07
ELI LILLY & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

A "probe" as used herein is a nucleic acid compound that hybridizes with another nucleic acid compound, and is useful for blot hybridizations, for example. A probe is at least 15 base pairs in length, its sequence being at least 90% identical with the nucleic acid molecules disclosed herein, or fragments thereof, or the complements thereof. A probe may or may not be labeled with a detectable moiety. As used herein, a probe is useful for hybridization analysis to identify sequences homologous to those disclosed herein.
The term "hybridization" as used herein refers to a process in which a single-stranded nucleic acid molecule joins with a complementary strand through nucleotide base pairing. "Selective hybridization" refers to hybridization under conditions of high stringency. The degree of hybridization depends upon, for example, the degree of complementarity, the stringency of hybridization, and the length of hybridizing strands.
The term "stringency" refers to hybridization conditions. High stringency conditions disfavor non-homologous basepairing. Low stringency conditions have the opposite effect. Stringency may be altered, for example, by temperature and salt concentration.
"Low stringency" conditions comprise, for example, a temperature of about 37.degree. C. or less, a formamide concentration of less than about 50%, and a moderate to low salt (SSC) concentration; or, alternatively, a temperature of about 50.degree. C. or less, and a moderate to high salt (SSPE) concentration.
"High stringency" conditions comprise a temperature of about 42.degree. C. or less, a formamide concentration of less than about 20%, and a low salt (SSC) concentration; or, alternatively, a temperature of about 65.degree. C., or less, and a low salt (SSPE) concentration.

Problems solved by technology

Moreover, anti-Candida compounds frequently do not possess clinically significant activity against other fungal species.
While compounds that target IPC synthase bode well for the future of anti-fungal therapy, presently there are no clinically useful compounds that act at this step.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of a DNA Vector for Expressing the C. glabrata IPC synthase Gene in Homologous or Heterologous Host

An expression vector suitable for expressing the IPC synthase gene of C. glabrata (SEQ ID NO:1) in E. coli contains an origin of replication (Ori), an ampicillin resistance gene (Amp) useful for selecting cells that have incorporated the vector following tranformation. The vector also includes the T7 promoter and T7 terminator sequences in operable linkage to the coding region of the IPC synthase gene. Parent plasmid pET11A (obtained from Novogen, Madison, Wisc.) is linearized by digestion with appropriate endonucleases and ligated to a DNA fragment comprising the coding region of the C. glabrata IPC synthase gene.

The IPC synthase gene ligated into the expression vector is modified at the 5' end (amino terminus of encoded protein) in order to simplify purification of the encoded IPC synthase protein product. For this purpose, an oligonucleotide encoding 8 histidine residue...

example 2

Expression of C. glabrata IPC synthase Gene in Echerichia coli and Purification of IPC synthase Enzyme

A plasmid from Example 1 is transformed into E. coli BL21 (DE3)(hsdS gal lcIts857 ind1Sam7nin5lacUV5-T7gene 1) using standard methods (See e.g. Sambrook et al. Supra). Transformants, selected for resistance to ampicillin, are chosen at random and tested for the presence of the vector by agarose gel electrophoresis using quick plasmid preparations. Colonies that contain the vector are grown, processed, and the protein encoded by the IPC synthase gene is purified by immobilized metal ion affinity chromatography (IMAC), essentially as described in U.S. Pat. No. 4,569,794, the entire contents of which is hereby incorporated by reference.

Briefly, the IMAC column is prepared as follows. A metal-free chelating resin (e.g. SEPHAROSE 6B IDA, Pharmacia) is washed in distilled water to remove preservative substances and infused with a suitable metal ion [e.g. Ni(II), Co(II), or Cu(II)] by addi...

example 3

Biochemical Assay for Inhibitors of IPC synthase Using Fungal Membrane Preparations

The activity of the IPC synthase enzyme is assayed by preparing membranes from C. glabrata, for example, and using said membranes as a source of IPC synthase activity.

A suitable, rich medium, for example YEPD, is innoculated with a culture and allowed to grow overnight at room temperature with vigorous shaking. About 250 ml of fresh medium containing 20 .mu.g / ml myo-inositol is innoculated with the overnight culture, and grown overnight at 30.degree. C. Cells are harvested by centrifugation and resuspended in ice cold 50 mM potassium phosphate buffer, pH 7. Cells are washed twice in the same buffer and then resuspended in the same buffer containing 5 mM dithiothreitol (DTT), 1 .mu.g / ml aprotinin, 0.6 .mu.M leupeptin, 1 mM PMSF, and 1 .mu.g / ml pepstatin A. Cells are ruptured using glass beads in a procedure that involves 5 successive vortexings each for 30 seconds followed by 2 to 5 minute intervals of...

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Abstract

The invention provides isolated nucleic acid compounds encoding IPC synthase or subunit thereof from fungal cells. Also provided are vectors and transformed heterologous host cells for expressing IPC synthase and a method for identifying compounds that inhibit a fungal IPC synthase.

Description

BACKGROUND OF THE INVENTIONThis invention relates to recombinant DNA technology. In particular the invention pertains to the isolation of novel genes and proteins that encode inositolphosphoryl ceramide synthase (IPC) synthase or subunit thereof from a variety of fungi and the use of said proteins in screens for inhibitors of IPC Synthase.The incidence of life-threatening fungal infections is increasing at an alarming rate. With the exception of Staphylococci infections, the fungus C. albicans represents the fastest growing area of concern in hospital acquired bloodstream infections. About 90% of nosocomial fungal infections are caused by species of Candida with the remaining 10% being attributable to infections by Aspergillus, Cryptococcus, and Pneumocystis. While effective antifungal compounds have been developed for Candida there is growing concern that the rise in the incidence of fungal infections may portend greater resistance and virulence in the future. Moreover, anti-Candid...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12N9/10C12N9/12
CPCC12N9/1258C12N9/1051C12N9/12C12N9/1205
Inventor HEIDLER, STEVEN ALANRADDING, JEFFREY ALAN
Owner ELI LILLY & CO
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