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Cloning, overexpression and therapeutic use of bioactive histidine ammonia lyase

a technology of histidine ammonia and lyase, which is applied in the field of bioactive, amino aciddegrading enzymes, can solve the problems of not being purified, many of the therapeutically beneficial properties associated with this hal were unknown, and the usefulness of l-histidinol as a single agent is limited

Inactive Publication Date: 2006-02-16
UNIVERSITY OF SOUTH CAROLINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Corynebacteriaceae HAL which Roberts et al. described was not in purified form.
As a result, many of the therapeutically beneficial properties associated with this HAL were unknown.
L-histidinol interferes with this conversion by inhibiting HDC.
L-histidinol has limited usefulness as a single agent due to its low half-life (Zaharko, D., Plowman, J., Ward, W., Dykes, D., and L. Malspeis, 1992.
Due to their enzymatic inhibition by histidinol, previously described histidine ammonia lyases have not been suitable candidates for use in combination therapies with these histidine analogs for treating pathologies such as cancer.
In addition to cancer, viral diseases such as Human Respiratory Syncytial Virus (RSV), Herpes Simplex Virus (HSV) and Human Immunodeficiency Virus (HIV), infect millions worldwide and cause major health problems.
This makes drug development against viruses very difficult.
However, such therapy is limited to combating only retroviruses.
But since these inhibitors target a specific enzyme, HIV protease, they cannot be effective against a wide range of viruses.
Although ganciclovir therapy may be beneficial to combating the Herpes Simplex Virus, it has limited or no application for treating other viruses.

Method used

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  • Cloning, overexpression and therapeutic use of bioactive histidine ammonia lyase
  • Cloning, overexpression and therapeutic use of bioactive histidine ammonia lyase
  • Cloning, overexpression and therapeutic use of bioactive histidine ammonia lyase

Examples

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working examples

Example 1

Isolation of the DNA Encoding HAL

[0120] The bacterium from the family Corynebacteriaceae that produces bioactive histidine ammonia lyase (HAL) was grown in 100 ml of Luria broth overnight at 30° C. The cells were harvested and resuspended in 10 ml of 50 mM Tris (pH 7.5) with 10 mM EDTA. Solid lysozyme was added to 0.2 mg / ml and the suspension was incubated at 4° C. for 30 minutes. Following this incubation, the suspension was frozen for several hours at −70° C. Upon thawing, SDS was added to 0.1% and proteinase K was added to 0.2 mg / ml and was incubated at 37° C. overnight. Next, RNAse was added to 0.1 mg / ml and the mixture incubated at 55° C. for 30 minutes. The resulting DNA was extracted five times with an equal volume of phenol / chloroform (1:1) and precipitated with 2 volumes of absolute ethanol. The DNA was spooled out on a glass Pasteur pipette, washed with ice cold 70% ethanol, and resuspended in a minimal amount of TE buffer.

[0121] Genomic DNA was restricted with...

example 2

Peptide Sequencing of HAL

[0132] HAL from a bacterium from the family Corynebacteriaceae that had been partially purified using ammonium sulfate and DEAE—Sephadex was resolved by SDS-PAGE. The separated material was electrophoretically transferred to Immobilon-P and stained with Coomassie Brilliant Blue. The major band of 55 000 daltons was excised and subjected to N-terminal sequencing. This fraction was sent to Commonwealth Biotechnologies, Inc. (Richmond, Va.), cleaved with BrCN, HPLC purified, and fractions sequenced. The data are shown in FIG. 2.

example 3

Expression of Histidine Ammonia Lyase from a Bacterium from the Family Corynebacteriaceae Using a High-Efficiency Prokaryotic Expression System

[0133] The E. coli expression plasmid pHUT102, depicted in FIG. 4, is designed to express HAL DNA sequences from the strong phage T7 gene 10 promoter using T7 RNA Polymerase. This vector, pSN75, is a derivative of pET11b (Novagen) that has an additional transcriptional terminator inserted upstream of the T7 promoter. This provides the target cassette in as transcriptionally silent a context as possible.

[0134] Two mutagenic oligonucleotides, TM96 and TM97 (see Table 1), were synthesized based on histidine ammonia lyase sequence. The N-terminal oligo adds an NdeI site at the ATG start codon and the C-terminal oligo adds a BamHI site just beyond the C-terminus. These were used to thermal cycle amplify the HAL gene out of the genome of a bacterium from the family Corynebacteriaceae. The resulting fragment was restricted with NdeI and BamHI and ...

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Abstract

Histidine ammonia lyase (HAL) isolated from Corynebacteriaceae can decrease serum histidine levels, induce accumulation of urocanic acid, and is not inhibited by L-histidinol. As a result, histidine ammonia lyases similar to the one isolated from Corynebacteriaceae are uniquely suitable for combination therapy with L-histidinol to treat histidine- and / or histamine-dependent pathologies, for example, infectious viruses, such as human Respiratory Syncytial Virus (RSV), Herpes Simplex Virus (HSV), and Human Immunodeficiency Virus (HIV), as well as cancers.

Description

This Application claims the benefit of provisional application 60 / 197,770, filed Apr. 14, 2000.FIELD OF THE INVENTION [0001] The present invention relates generally to bioactive, amino acid-degrading enzymes, more specifically to a histidine ammonia lyase derived from a bacterium belonging to the family Corynebacteriaceae, and to conservative variants thereto. Also described is the use of histidine ammonia lyase, singly or combined with L-histidinol, for treating various viral diseases. BACKGROUND OF THE INVENTION [0002] Histidine ammonia lyase (EC 4.3.1.3) catalyzes the conversion of L-histidine to urocanic acid and ammonia. This is the first step in the degradation of histidine in both mammals and bacteria. A deficiency in this enzyme results in histidinemia, which is characterized by high serum histidine levels. [0003] An isolated histidine ammonia lyase enzyme is one agent for treating increased histidine levels. Several lines of evidence indicate that in vivo depletion of serum...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/53A61K31/4172C12N15/09A61K31/417A61K35/76A61K38/00A61K38/51A61K45/00A61K48/00A61P31/12A61P31/14A61P31/18A61P31/20A61P31/22A61P35/00A61P37/06C12N9/88C12N15/60
CPCA61K38/00C12Y403/01003C12N9/88A61K48/00A61P31/12A61P31/14A61P31/18A61P31/20A61P31/22A61P35/00A61P37/06Y02A50/30
Inventor ROBERTS, JOSEPHSETHURAMAN, NATARAJANMACALLISTER, THOMAS
Owner UNIVERSITY OF SOUTH CAROLINA
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