title: Identification of the minimal region in lipase ABC transporter recognition domain of Pseudomonas fluorescens for secretion and fluorescence of green fluorescent protein.
abstract: BACKGROUND
  TliA is a thermostable lipase secreted by the type 1 secretion system (T1SS) of Pseudomonas fluorescens. The secretion is promoted by its secretion/chaperone domain located near the C-terminus, which is composed mainly of four Repeat-in-Toxin (RTX) repeats. In order to identify the minimal region of TliA responsible for its secretion, five different copies of the secretion/chaperone domain, each involving truncated N-terminal residues and a common C-terminus, were acquired and named as lipase ABC transporter recognition domains (LARDs). Each LARD was fused to epidermal growth factor (EGF) or green fluorescent protein (GFP), and the secretion of EGF-LARD or GFP-LARD fusion proteins was assessed in Escherichia coli with ABC transporter.
  
  
  RESULTS
  Among the fusion proteins, GFP or EGF with 105-residue LARD3 was most efficiently secreted. In addition, GFP-LARD3 emitted wild type GFP fluorescence. Structurally, LARD3 had the 4 RTX repeats exposed at the N-terminus, while other LARDs had additional residues prior to them or missed some of the RTX repeats. LARD3 was both necessary and sufficient for efficient secretion and maintenance of GFP fluorescence in E. coli, which was also confirmed in P. fluorescens and P. fluorescens ▵tliA, a knock-out mutant of tliA.
  
  
  CONCLUSIONS
  LARD3 was a potent secretion signal in T1SS for its fusion flanking RTX motif, which enhanced secretion and preserved the fluorescence of GFP. LARD3-mediated secretion in E. coli or P. fluorescens will enable the development of enhanced protein manufacturing factory and recombinant microbe secreting protein of interest in situ.
authors.affiliation: Korea Science Academy of KAIST, 899 Tanggam 3-Dong, Busanjin-Gu, Busan, 614-822, Korea.
authors.forename: Yeonwoo
authors.initials: Y
authors.lastname: Park
authors.forename: Yuseok
authors.initials: Y
authors.lastname: Moon
authors.forename: Jungmin
authors.initials: J
authors.lastname: Ryoo
authors.forename: Nayeon
authors.initials: N
authors.lastname: Kim
authors.forename: Hyounghoon
authors.initials: H
authors.lastname: Cho
authors.forename: Jung Hoon
authors.initials: JH
authors.lastname: Ahn
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D058947:Bacterial Secretion Systems; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011551:Pseudomonas fluorescens / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D049452:Green Fluorescent Proteins; D008049:Lipase