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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2017, Vol. 37 Issue (2): 81-87    DOI: 10.13523/j.cb.20170212
    
Development of Two Recombinant Enzymes: Cystathionine β-synthase and Cystathionine β-lyase Vital for Enzymatic Cycling Homocysteine Assay and Preliminary Characterization of the Corresponding Assay Kit
WANG Du-qiang1,2, SHEN Yun-long1, LIAO Yuan-ping1, LI De-bin1, LIU Hong-jun1, CHEN Shuai1, LU Da-ru2, ZHU Hua-xing1
1. R&D Department, Novoprotein Scientific (Wujiang) Inc, Suzhou 215222, China;
2. State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China
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Abstract  

The target genes of cystathionine β-synthase (CBS) and cystathionine β-lyase (CBL) from Saccharomyces cerevisiae were amplified by PCR and cloned into the expression vector pET28a respectively. The resulting plasmids were then transformed to E.coli BL21(DE3) cells. After induction of the gene expression with IPTG and protein purification, the purities of recombinant CBS and CBL were both near 90% with a final yield of 80%. The soluble expression levels of CBS and CBL were 26 mg/L and 332 mg/L respectively, and the specific activities of purified enzymes were determined to be 15 U/mg and 72 U/mg. With utilization of recombinant CBS and CBL as two key components, an enzymatic cycling homocysteine (Hcy) assay kit was developed and characterized preliminarily, whose functionality and stability are believed to meet the requirements of in vitro diagnostic usage. In addition, functionality comparison of our Hcy assay kit with the imported product that has been sold on the market did not show any obvious difference.

Key words： Cystathionine β-lyase      Homocysteine      Cystathionine β-synthase      Enzymatic activity measurement     
Received: 15 August 2016      Published: 25 February 2017
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WANG Du-qiang, SHEN Yun-long, LIAO Yuan-ping, LI De-bin, LIU Hong-jun, CHEN Shuai, LU Da-ru, ZHU Hua-xing. Development of Two Recombinant Enzymes: Cystathionine β-synthase and Cystathionine β-lyase Vital for Enzymatic Cycling Homocysteine Assay and Preliminary Characterization of the Corresponding Assay Kit. China Biotechnology, 2017, 37(2): 81-87.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20170212     OR     https://manu60.magtech.com.cn/biotech/Y2017/V37/I2/81

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