title: A vector system for ABC transporter-mediated secretion and purification of recombinant proteins in Pseudomonas species.
abstract: Pseudomonas fluorescens is an efficient platform for recombinant protein production. P. fluorescens has an ABC transporter secreting endogenous thermostable lipase (TliA) and protease, which can be exploited to transport recombinant proteins across the cell membrane. In this study, the expression vector pDART was constructed by inserting tliDEF, genes encoding the ABC transporter, along with the construct of the lipase ABC transporter recognition domain (LARD), into pDSK519, a widely used shuttle vector. When the gene for the target protein was inserted into the vector, the C-terminally fused LARD allowed it to be secreted through the ABC transporter into the extracellular medium. After secretion of the fused target protein, the LARD containing a hydrophobic C terminus enabled its purification through hydrophobic interaction chromatography (HIC) using a methyl-Sepharose column. Alkaline phosphatase (AP) and green fluorescent protein (GFP) were used to validate the expression, export, and purification of target proteins by the pDART system. Both proteins were secreted into the extracellular medium in P. fluorescens. In particular, AP was secreted in several Pseudomonas species with its enzymatic activity in extracellular media. Furthermore, purification of the target protein using HIC yielded some degree of AP and GFP purification, where AP was purified to almost a single product. The pDART system will provide greater convenience for the secretory production and purification of recombinant proteins in Gram-negative bacteria, such as Pseudomonas species.
authors.affiliation: Korea Science Academy of KAIST, Busanjin-Gu, Busan, South Korea.
authors.forename: Jaewook
authors.initials: J
authors.lastname: Ryu
authors.affiliation: Korea Science Academy of KAIST, Busanjin-Gu, Busan, South Korea.
authors.forename: Ukjin
authors.initials: U
authors.lastname: Lee
authors.affiliation: Korea Science Academy of KAIST, Busanjin-Gu, Busan, South Korea.
authors.forename: Jiye
authors.initials: J
authors.lastname: Park
authors.affiliation: Korea Science Academy of KAIST, Busanjin-Gu, Busan, South Korea.
authors.forename: Do-Hyun
authors.initials: DH
authors.lastname: Yoo
authors.affiliation: Korea Science Academy of KAIST, Busanjin-Gu, Busan, South Korea hoony@kaist.ac.kr.
authors.forename: Jung Hoon
authors.initials: JH
authors.lastname: Ahn
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D008969:Molecular Sequence Data; D011549:Pseudomonas / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D017422:Sequence Analysis, DNA
chemical_list: D018528:ATP-Binding Cassette Transporters; D004269:DNA, Bacterial; D011994:Recombinant Proteins