High Expression,Purification and Characterization of cDNA Encoding M3-M4 Loop in the Essential Subuint of hNMDA Receptor
ZHANG Yu mei 1) , SUN Chang kai 1) 2) 3) , FAN Ming 2) , LI Wu ju 2) , LIU Shu hong 2) ,ZHAO Jie 1) , HAN Da yue 3) , WANG Jia xi 2)
( 1) Institute for Brain Disorders, Dalian Medical University, Dalian 116027, China; 2) Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing 100850, China; 3) Department of Neurology, Chinese PLA 210 Hospital, Dalian 116021, China
Abstract：A cDNA fragment encoding M3 M4 loop in the essential subuint of human N \|methyl\| D \|aspartate(NMDA) receptor was obtained by RT PCR using the total RNA extracted from a human brain glioma as template. The cDNA fragment was structurally optimized by means of CAD and then inserted into the high expression vector pBV220. The resultant recombinant plasmid which was confirmed by DNA sequencing was introduced into the competent cell of E.coli DH5α and inducibly expressed by means of shifting the culture temperature from 30℃ to 42℃. The recombinant protein obtained was assayed and purified by the preparative PAGE, and then characterized by a series method of identification, such as apparent molecular weight, immunoreactivity, and peptide fingerprinting. As a result, a prokaryotic expression vector for M3 M4 loop in the essential subuint of human NMDA receptor was constructed and named as pBV NR1L3, the high expression of which was achieved by structure optimization. The recombinant protein accounted for 29% of the total bacterial protein, and was purified to a purity of 95%.
张玉梅,孙长凯,范明,李伍举,刘淑红,赵杰,韩大跃,王嘉玺. 人NMDA受体主亚基M3-M4环基因片段的高效表达、纯化与鉴定[J]. 中国生物化学与分子生物学报, 2003, 19(05): 588-593.
ZHANG Yu mei 1) , SUN Chang kai 1) 2) 3) , FAN Ming 2) , LI Wu ju 2) , LIU Shu hong 2) ,ZHAO Jie 1) , HAN Da yue 3) , WANG Jia xi 2). High Expression,Purification and Characterization of cDNA Encoding M3-M4 Loop in the Essential Subuint of hNMDA Receptor. Chinese Journal of Biochemistry and Molecular Biol, 2003, 19(05): 588-593.
