title: Strategy for successful expression of the Pseudomonas putida nitrile hydratase activator P14K in Escherichia coli.
abstract: BACKGROUND
  Activators of Nitrile hydratase (NHase) are essential for functional NHase biosynthesis. However, the activator P14K in P. putida is difficult to heterogeneously express, which retards the clarification of the mechanism of P14K involved in the maturation of NHase. Although a strep tag containing P14K (strep-P14K) was over-expressed, its low expression level and low stability affect the further analysis.
  
  
  RESULTS
  We successfully expressed P14K through genetic modifications according to N-end rule and analyzed the mechanism for its difficult expression. We found that mutation of the second N-terminal amino-acid of the protein from lysine to alanine or truncating the N-terminal 16 amino-acid sequence resulted in successful expression of P14K. Moreover, fusion of a pelB leader and strep tag together (pelB-strep-P14K) at the N-terminus increased P14K expression. In addition, the pelB-strep-P14K was more stable than the strep-P14K.
  
  
  CONCLUSIONS
  Our results are not only useful for clarification of the role of P14K involved in the NHase maturation, but also helpful for heterologous expression of other difficult expression proteins.
authors.affiliation: Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Peoples Republic of China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Liu
authors.forename: Wenjing
authors.initials: W
authors.lastname: Cui
authors.forename: Yueqin
authors.initials: Y
authors.lastname: Fang
authors.forename: Yuechun
authors.initials: Y
authors.lastname: Yu
authors.forename: Youtian
authors.initials: Y
authors.lastname: Cui
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Xia
authors.forename: Michihiko
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Zhemin
authors.initials: Z
authors.lastname: Zhou
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D006836:Hydro-Lyases / Q000378:metabolism*; D008969:Molecular Sequence Data; D055550:Protein Stability; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D006836:Hydro-Lyases; C048831:nitrile hydratase