Enzymatic cycling assays for homocysteine and cystathionine

A technology of homocysteine ​​and cystathionine, applied in the field of enzymatic cycle determination of homocysteine ​​and cystathionine, can solve the problems such as unreported enzymatic cycle assay

Inactive Publication Date: 2005-05-04
AXIS SHIELD DIAGNOSTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

No enzymatic cycling assays have been ...

Method used

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  • Enzymatic cycling assays for homocysteine and cystathionine
  • Enzymatic cycling assays for homocysteine and cystathionine
  • Enzymatic cycling assays for homocysteine and cystathionine

Examples

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Embodiment 1

[0100] Cloning and expression of cystathionine β-lyase gene from Escherichia coli

[0101] In this example, the E. coli CBL gene was cloned using standard PCR procedures. The isolated gene is inserted into an expression vector with an affinity marker sequence (6HIS), and the expressed protein is purified with an affinity column. Large amounts of CBL enzyme activity in host cells were transformed with expression vector constructs and purified from cell lysates.

[0102] Cloning of Escherichia coli cystathionine β-lyase (CBL, EC 4.4.1.8) gene

[0103] A standard PCR procedure used the Extended High-Fidelity PCR System (Roche Biochemicals) for amplification of the E. coli CBL gene, with ONE SHOT TOP10 competent E. coli cells (Invitrogen) as the source of genomic DNA. The PCR primer sequences used for CBL cloning were as follows: (CBL N-terminal primer: 5'-CGACGGATCCGATGGCGGACAAAAAGCTTG-3'; SEQ ID NO: 1) and (CBL C-terminal primer: 5'-CGCAGCAGCTGTTATACAATTCGCGCAAA-3'; SEQ ID N...

Embodiment 2

[0110] Cloning and Expression of Yeast Cystathionine β-Synthase (CBS) Gene in Yeast

[0111] In this example, the PCR method was used to clone the CBS gene from yeast. The PCR fragment containing the CBS gene was inserted into the expression vector with and without an affinity tag, and the addition of the tag facilitated the purification of the expressed gene product.

[0112] A laboratory strain of S. cerevisiae (5153-11-1 (his3 met2)) was used as the source of the CBS gene. Cells were grown on YEPD at 32°C, centrifuged, washed with water and transferred to 200 μl of yeast plasmid buffer (100 mM NaCl, 10 mM Tris (pH 8), 1 mM EDTA, 0.2% SDS). The cell suspension was mixed with an equal volume of 0.45 mm glass beads and vortexed at high speed for 3 x 30 seconds. Liquid was removed by pipetting and centrifuged in a microfuge for 2 minutes to remove cell debris. The supernatant was extracted with PCI (phenol:chloroform:isoamyl alcohol (25:24:1) and chloroform. Sodium chlorid...

Embodiment 3

[0123] Cloning, expression and purification of yeast cystathionine β-synthase (CBS) gene in Escherichia coli

[0124] In this example, standard PCR and molecular biology techniques were used to clone the Saccharomyces cerevisiae (INVSc1) cystathionine β-synthase (CBS) gene as a fusion with an amino-terminal glutathione-S-transferase (GST). protein. Addition of this glutathione-S-transferase domain allows for a single-step affinity purification of the CBS protein from E. coli culture medium and also assists in the solubilization of the active form of the enzyme and / or facilitates proper refolding.

[0125] clone

[0126] A laboratory strain of S. cerevisiae (INVSc1 ) (Invitrogen) was used as the source of the CBS gene. Single colonies of INVSc1 yeast were boiled in 100 μl deionized water for 5 min and vortexed rapidly for 2 min in the presence of an equal volume of 0.45 mm glass beads. One microliter of this cell lysate was then used as a source of DNA for PCR amplificatio...

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Abstract

The present invention provides an enzymatic cycling assay for assessing the amount of homocysteine and/or cystathionine in a solution such as blood, blood derivatives, or urine. The assay comprises the steps of contacting the solution containing homocysteine and/or cystathionine to form a reaction mixture, with CBS, or a derivative thereof, L-serine, and CBL, or a derivative thereof, for a time period sufficient to catalyze the cyclical conversion of homocysteine form to cystathionine and the reconversion of cystathionine to homocysteine with the production of pyruvate and ammonia; determining the amount of homocysteine and/or ammonia present in the reaction mixture; and determining the amount of homocysteine and/or cystathionine present in the solution based on the amount of pyruvate and/or ammonia formed. Expression vectors and isolation procedures for CBS, or derivatives thereof, and CBL, or derivatives thereof, are also provided as well as test kits for carrying out the assay. In preferred embodiments, the assays can be conducted in 15 minutes or less, with a minimum of enzyme usage.

Description

[0001] Related Patent Applications [0002] This application is a continuation-in-part of U.S. Patent Application Serial No. 09 / 704,036, filed November 1, 2000, which claims Provisional Patent Application Serial No. 60 / 163,126, filed November 2, 1999 and May 10, 2000 Serial No. 60 / 203,349 filed on the 1st date, the contents and teachings of each of the foregoing applications are incorporated herein by reference. [0003] sequence list [0004] A sequence listing of 21 sequences pertaining to the present invention is incorporated herein by reference as a computer readable ASCII file and attached with two (2) original CDs under 37CFR 1.821(c), which are identical under 37CFR 1.821(e) copy and one (1) identical copy thereof pursuant to 37 CFR 1.52(e). background of the invention [0005] High levels of homocysteine ​​in human plasma are associated with an increased risk of coronary heart disease, stroke, arteriosclerosis an...

Claims

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Application Information

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IPC IPC(8): C12Q1/00C12N9/00C12N15/52C12Q1/26C12Q1/32C12Q1/527G01N33/53G01N33/68
CPCG01N33/6815C12Q1/26C12Q1/32Y10S435/975Y10S435/968C12Q1/527
Inventor H·K·韦伯J·欧文斯R·里德克D·弗里斯特M·莱加兹G·川崎S·罗森
Owner AXIS SHIELD DIAGNOSTICS
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