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Fermentation and purification of actinomadura chromoprotein and related species

A technology of actinomycetes madura and chromoproteins, applied in fermentation, methods using bacteria, methods based on microorganisms, etc., can solve the problems of reduced toxicity and increased stability, and achieve reduced toxicity, increased stability, and high efficacy increased effect

Inactive Publication Date: 2009-07-15
WYETH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Furthermore, said alteration of the apoprotein may result in, for example, a chromoprotein with reduced toxicity or a chromophore with increased potency or stability.

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  • Fermentation and purification of actinomadura chromoprotein and related species
  • Fermentation and purification of actinomadura chromoprotein and related species
  • Fermentation and purification of actinomadura chromoprotein and related species

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[0188] It is understood and anticipated that variations on the principles of the invention may be effected by persons skilled in the art and that such modifications are intended to be included within the scope of the invention.

[0189] The following examples of the invention are set forth to further illustrate the invention and should not be construed as limiting the invention in any way.

example 1

[0191] Separation and characterization of chromoprotein and apoprotein

[0192] Example 1 - Seed Culture

[0193] Actinomyces madurai 21G792 was preserved as frozen whole cells (frozen vegetative mycelium, FVM) prepared from cells grown in ATCC medium 172 for 72 hours (dextrose 1%, soluble starch 2%, yeast extract 0.5%, and N-Z amine type A 0.5%, CaCO 3 0.1% pH 7.3). Glycerol was added to 20% and cells were frozen at -150°C.

[0194] A seed medium containing pH 6.9 was prepared containing: 1.0% dextrose, 2.0% soluble starch, 0.5% yeast extract, 0.5% N-Z amine type A (Sheffield), and 0.1% CaCO 3 . In a 25mm x 150mm glass culture tube, inoculate 7ml of seed medium and two glass beads. Sufficient inoculum from the agar culture was used to provide mixed seeds after 72 hours of incubation. The primary seed tubes were incubated for 72 hours at 28°C, 250 rpm using a gyro spinner flask with a 2 inch eccentricity. Primary seeds (approximately 14% inoculum) were then used to in...

example 2

[0196] Example 2 - Fermentation

[0197] Prepare a ferment production medium at pH 6.9 containing: 2.0% sucrose, 0.5% molasses, 0.5% CaCO 3 , 0.2% peptone, 0.002% magnesium sulfate-7H 2 O, 0.001% ferrous sulfate-7H 2 O, 0.05% sodium bromide, and 0.2% sodium acetate. Sixty 250ml Erlenmeyer flasks each with 50ml of ferment production medium were inoculated with 2ml (4.0%) of secondary seed ferment and gyroscoped at 28°C, 250rpm (2" eccentric distance) Cultivation is carried out.The fermentation is then continued for about 72 to 96 hours and harvested for further processing.

[0198] The combined whole broth (60x50ml) was centrifuged at 3800 rpm for 30 minutes. The supernatant was then lyophilized and the residual powder was suspended in a small volume (eg, 300 ml) of H 2 O middle. After centrifugation, the brown solution was then loaded at 4 °C in the dark with 2 6L Sephadex G75 glass column in O. Fractions collected were each 40 ml and tested in a Biochemical Induction ...

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Abstract

The present invention provides methods for production and purification of active chromoproteins produced by Actinomadura sp. 21G792. The chromoproteins are useful for developing pharmaceutical compositions and treating diseases such as cancer or bacterial infections.

Description

[0001] sequence listing [0002] This application includes a Sequence Listing setting forth the nucleic acid and amino acid sequences discussed herein. technical field [0003] The present invention relates to a method for producing and purifying active chromoprotein produced by Actinomadura sp 21G792. The chromoproteins can be used in the development of pharmaceutical compositions and in the treatment of diseases such as cancer or bacterial infection. Background technique [0004] Enediynes are potent cytotoxic polyketide species produced by members of the Actinomycetales that have been used in the treatment of cancer. The typical mode of action of enediyne drugs is through single- and double-stranded DNA cleavage. DNA cleavage is induced by hydrogen abstraction from the deoxyribose sugar backbone by diradicals generated by Bergman-type ring aromatization from enediyne rings. There are currently two enediynes approved clinically for the treatment of cancer: calicheamici...

Claims

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Application Information

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IPC IPC(8): A61K47/48C07K14/36C07D491/08
CPCC12N1/20A61K47/48561A61K47/48569C12R1/03A61K47/48484C07K14/36C07D413/12C12P17/16A61K47/6829A61K47/6849A61K47/6851A61P31/04A61P35/00C12N1/205C12R2001/03
Inventor 尤金·约瑟夫·维杜纳斯蔡国真蔡平贾斯廷·基思·莫兰帕梅拉·休·芬克·沙博诺洛德斯·珍妮特·戈登
Owner WYETH LLC
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