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KMID : 0613820110210010119
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Journal of Life Science
2011 Volume.21 No. 1 p.119 ~ p.126
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Overexpression and Activity Analysis of Cystathionine γ-Lyase Responsible for the Biogenesis of H₂S Neurotransmitter
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Kim Kyoung-Ran
Byun Hae-Jung
Cho Hyun-Nam
Kim Jung-Hyun
Yang Seun-Ah
Jhee Kwang-Hwan
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Abstract
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There is a growing recognition of the significance of H2S as a biological signaling molecule involved in vascular and nervous system functions. In mammals, two enzymes in the transsulfuration pathway, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL), are believed to be chiefly responsible for H₂S biogenesis. Genetic inborn error of CGL leads to human genetic disease, cystathioninuria, by accumulating cystathionine in the body. This disease is secondarily associated with a wide range of diseases including diabetes insipidus and Down"s syndrome. Although the human CGL (hCGL) overexpression is essential for the investigation of its function, structure, reaction specificity, substrate specificity, and protein-protein interactions, there is no clear report concerning optimum overexpression conditions. In this study, we report a detailed analysis of the overexpression conditions of the hCGL using a bacterial system. Maximum overexpression was obtained in conditions of low culture temperature after inducer addition, performing low aeration during overexpression, and using a low concentration inducer (0.1 mM, IPTG) for induction. Expressed hCGL was purified by His-tag affinity column chromatography and confirmed by Western blot using hCGL antibody and enzyme activity analysis. We also report that the His tag with TEV site attached protein exhibits 76% activity for α-γ elimination reaction with L-cystathionine and 88% for α-β elimination reaction with L-cysteine compared to those of wild type hCGL, respectively. His tag with TEV site attached protein also exhibits a 420 ㎚ absorption maximum, which is attributed to the binding cofactor, pyridoxal 5’-phosphate (PLP).
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KEYWORD
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Overexpression, cystathionine γ-lyase, cystathioninuria, homocysteine, cysteine
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