title: [Homologous cloning and expression of PDS gene from Dunaliella salina in Escherichia coli].
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.
  
  
  CONCLUSIONS
  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.
authors.forename: Feng
authors.initials: F
authors.lastname: Zhou
authors.forename: Fei
authors.initials: F
authors.lastname: Huang
authors.forename: Linhan
authors.initials: L
authors.lastname: Bai
mesh_terms: D000460:Chlorophyta / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism
chemical_list: D010088:Oxidoreductases; C022398:phytoene dehydrogenase