Key Laboratory of Bio-resources and Eco-environment of Ministry of Education,College of Life Sciences,Sichuan University,Chengdu 610064,Sichuan Province,China 在期刊界中查找 在百度中查找 在本站中查找
Key Laboratory of Bio-resources and Eco-environment of Ministry of Education,College of Life Sciences,Sichuan University,Chengdu 610064,Sichuan Province,China 在期刊界中查找 在百度中查找 在本站中查找
Key Laboratory of Bio-resources and Eco-environment of Ministry of Education,College of Life Sciences,Sichuan University,Chengdu 610064,Sichuan Province,China 在期刊界中查找 在百度中查找 在本站中查找
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Supported by the National Natural Science Foundation of China (30970043)
Abstract:[Objective]Phytoene desaturase PDS is a eukaryotic nuclear membrane binding protein,we used different expression methods to search for the soluble expression strategy of membrane binding protein in Escherichia coli.[Methods]We cloned the full-length cDNA sequence of PDS from Dunaliella salina through RACE.First,we utilized prokaryotic expression vector pET-28a to construct pET-28a-PDS vector.Then,we substituted PLtac promoter for T7 promoter in pET-28a to construct pET-PLtac-PDS vector.Last,we constructed pET-Mistic-PDS fusion vector by integrating Mistic sequence into pET-28a. All were transformed into BL21 (DE3) for protein expression.[Results]The 2237-bp full-length cDNA sequence of PDS was cloned,including a 1749-bp open reading frame,encoding 582 amino acids (NCBI accession: GQ923693.1).The expression of PDS protein was low via pET-28a-PDS and pET-PLtac-PDS vector,and proteins were mostly expressed in inclusion body.The expression of PDS protein was significantly increased via pET-Mistic-PDS vector,in addition most were expressed as soluble protein which possessed dehydrogenase activity.[Conclusion]Mistic as the solubilization label was able to promote proper folding of membrane proteins and improve solubility. Protease activity assay proved that Mistic could maintain the enzyme activity.