title: [High expression of agarase AgaD in Escherichia coli].
abstract: OBJECTIVE
  We constructed highly efficient expression systems for agarase AgaD and optimized its culture conditions.
  
  
  METHODS
  First, the codon usage of AgaD was optimized to make it suitable for expression in E. coli. Then, the gene expression vector was transformed into different E. coli hosts. According to the "N-end rule" that is related to the in vivo half-life of a protein, a mutant was constructed. Finally, the effects of CaCl2 and glycine on enzyme production were evaluated.
  
  
  RESULTS
  A highly efficient expression system of agarase AgaD was constructed, named pET-22b (+)-optagaDx-AD494 ( E3). Replacing N-terminal second amino acid phenylalanine with alanine significantly improved agarase production and shortened the fermentation period. The extracellular enzyme activity was further up-regulated by CaCll and glycine. After optimization, the extracellular enzyme production raised from 20 U/L to 11300 U/L, more than 500 folds.
  
  
  CONCLUSIONS
  The high expression system of AgaD provides good basis for further studying agarases.
authors.forename: Huan
authors.initials: H
authors.lastname: Liu
authors.forename: Weibin
authors.initials: W
authors.lastname: Zhang
authors.forename: Dan
authors.initials: D
authors.lastname: Liu
authors.forename: Wengong
authors.initials: W
authors.lastname: Yu
authors.forename: Xinzhi
authors.initials: X
authors.lastname: Lu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering
chemical_list: D001426:Bacterial Proteins; D006026:Glycoside Hydrolases; C019624:agarase