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
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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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