A Vector System for ABC Transporter-Mediated Secretion and Purification of Recombinant Proteins in Pseudomonas Species

Cited 4 time in webofscience Cited 0 time in scopus
  • Hit : 239
  • Download : 0
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.
Publisher
AMER SOC MICROBIOLOGY
Issue Date
2015-03
Language
English
Article Type
Article
Keywords

GRAM-NEGATIVE BACTERIA; ESCHERICHIA-COLI; SERRATIA-MARCESCENS; CRYSTAL-STRUCTURE; I SECRETION; ALKALINE-PHOSPHATASE; RECOGNITION DOMAIN; FLUORESCENS SBW25; LIPASE SECRETION; IDENTIFICATION

Citation

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, v.81, no.5, pp.1735 - 1744

ISSN
0099-2240
DOI
10.1128/AEM.03514-14
URI
http://hdl.handle.net/10203/195779
Appears in Collection
RIMS Journal Papers
Files in This Item
There are no files associated with this item.
This item is cited by other documents in WoS
⊙ Detail Information in WoSⓡ Click to see webofscience_button
⊙ Cited 4 items in WoS Click to see citing articles in records_button

qr_code

  • mendeley

    citeulike


rss_1.0 rss_2.0 atom_1.0