title: Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD).
abstract: BACKGROUND
  ATP binding cassette (ABC) transporter secretes the protein through inner and outer membranes simultaneously in gram negative bacteria. Thermostable lipase (TliA) of Pseudomonas fluorescens SIK W1 is secreted through the ABC transporter. TliA has four glycine-rich repeats (GGXGXD) in its C-terminus, which appear in many ABC transporter-secreted proteins. From a homology model of TliA derived from the structure of P. aeruginosa alkaline protease (AprA), lipase ABC transporter domains (LARDs) were designed for the secretion of fusion proteins.
  
  
  RESULTS
  The LARDs included four glycine-rich repeats comprising a beta-roll structure, and were added to the C-terminus of test proteins. Either Pro-Gly linker or Factor Xa site was added between fusion proteins and LARDs. We attached different length of LARDs such as LARD0, LARD1 or whole TliA (the longest LARD) to three types of proteins; green fluorescent protein (GFP), epidermal growth factor (EGF) and cytoplasmic transduction peptide (CTP). These fusion proteins were expressed in Escherichia coli together with ABC transporter of either P. fluorescens or Erwinia chrysanthemi. Export of fusion proteins with the whole TliA through the ABC transporter was evident on the basis of lipase enzymatic activity. Upon supplementation of E. coli with ABC transporter, GFP-LARDs and EGF-LARDs were excreted into the culture supernatant.
  
  
  CONCLUSIONS
  The LARDs or whole TliA were attached to C-termini of model proteins and enabled the export of the model proteins such as GFP and EGF in E. coli supplemented with ABC transporter. These results open the possibility for the extracellular production of recombinant proteins in Pseudomonas using LARDs or TliA as a C-terminal signal sequence.
authors.affiliation: Korea Science Academy, #899, Tanggam 3-Dong, Busanjin-Gu, Busan, 614-822, Korea. hoony@kaist.ac.kr.
authors.forename: Chan Woo
authors.initials: CW
authors.lastname: Chung
authors.forename: Jinsun
authors.initials: J
authors.lastname: You
authors.forename: Kyeongyeon
authors.initials: K
authors.lastname: Kim
authors.forename: Yuseok
authors.initials: Y
authors.lastname: Moon
authors.forename: Hoeon
authors.initials: H
authors.lastname: Kim
authors.forename: Jung Hoon
authors.initials: JH
authors.lastname: Ahn