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.T he mass production of recombinant proteins continues to be an important issue in various industries (1, 2). In conventional methods, recombinant proteins are synthesized in prokaryotic cells, such as Escherichia coli, and are purified from the cell extract by biochemical means after cell lysis. A protein-manufacturing system that can simultaneously express and secrete recombinant proteins is far more efficient than conventional methods due to the reduced requirement for expensive extraction and purification procedures (3-6). Several features of Pseudomonas fluorescens, a Gram-negative psychrotrophic bacterium, make it one of the most useful organisms for recombinant protein production (7). P. fluorescens, living on the surfaces of most plants, is generally accepted as safe, since it has been consumed by humans for a long time (8). The safety of the ␣-amylase enzyme produced using P. fluorescens has been verified by pharmacological and toxicological studies in mice and rats (9). In addition to its biological safety, P. fluorescens is capable of withstanding various fermentation conditions in high-cell-density cultures and therefore can produce large quantities of recombinant proteins (10, 11). Several secretion systems exist natively in P. fluorescens, ranging from a type I secretion system (T1SS) to a type VI secretion system (T6SS) (12, 13). In particular, P. fluorescens has a type I secretion system that transports thermostable lipase, TliA, through the ATP-bin...

A lower isoelectric point increases signal sequence–mediated secretion of recombinant proteins through a bacterial ABC transporter

“…2A and supplemental Table S1). Similarly intriguing, other reports of recombinant protein secretion through ABC transporters often, if not always, involve acidic, negatively-charged target proteins: mannanase (13), phospholipase A 1 , 3 alkaline phosphatase (11), and epidermal growth factor (10). We also added the pI values of these proteins in Fig.…”

“…Plasmid pDART was used for the secretory production of different proteins (11). Plasmid vectors pFD10 and pBD10 were derivatives of pDART, constructed by adding codons for 10 aspartic acid residues to the target proteins in either the upstream or downstream position of MCS.…”

“…LARD3 (11). The pDART plasmid has a multiple-cloning site (MCS) directly followed by an in-frame LARD3 gene.…”

BIOMOSAIC Film: Artificial Biofilms with Catalytic and Self‐Sealing Properties

“…Simple addition of AP‐secreting microbial cells generated the phosphatase activity incorporated BIOMOSAIC films. The observed dephosphorylation from para ‐nitrophenyl phosphate was to produce 160 × 10 −6 m of para ‐nitrophenol in 24 h (Figure f) . The reusability of BIOMOSAIC–AP film tested by 3 consecutive times of the film incubation was that about 50.6 ± 0.5% activity was remained (Figure S13, Supporting Information).…”

“…Phosphate Hydrolysis Using BIOMOSAIC–AP Film : The biocatalytic activity of the BIOMOSAIC–AP film was determined using para ‐nitrophenyl phosphate ( p ‐NPP) as a substrate . The BIOMOSAIC–AP film (3.5 cm × 3.5 cm) was incubated with 25 mL of 5 × 10 −3 m p ‐NPP in a 0.1 m glycine buffer 100 × 10 −3 m Tris‐HCl (pH 8), 100 × 10 −3 m NaCl, and a 5 × 10 −3 m MgCl 2 buffer.…”