Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD)

Chung, Chan Woo; You, Jinsun; Kim, Kyeongyeon; Moon, Yuseok; Kim, Hoeon; Ahn, Jung Hoon

doi:10.1186/1475-2859-8-11

Cited by 23 publications

(22 citation statements)

References 50 publications

(59 reference statements)

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“…In this study, the export of recombinant human EGF attached to the C-terminal fragment (residues 302 to 476) of thermostable lipase (TliA) of Pseudomonas fluorescens SIK W1 is reported. These fragments, defined in previous work as a lipase ABC transporter recognition domain (LARD) (8), are secretion signals derived from TliA. Among the different LARDs, LARD3 (residues 372 to 476) was attached to the C terminus of human EGF.…”

Section: Methodsmentioning

confidence: 99%

“…Together with this plasmid, the ABC transporter gene, including PrtDEF, was introduced for the secretion of LARD3-linked recombinant EGF protein. The PrtDEF transporter of Erwinia chrysanthemi functions at 37°C, the optimum growth temperature for E. coli, while the Pseudomonas fluorescens TliDEF transporter must be expressed at 25 to 27°C for its proper function (1,8). E. coli Nissle 1917 or E. coli XL1-Blue was transformed with plasmids of EGF and the ABC transporter (Prt-DEF) and were designated Nissle-AC and XL1-AC, respectively (Table 2).…”

Section: Expression Of Human Egf In E Coli Nissle 1917mentioning

confidence: 99%

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Enhanced Wound Healing by Recombinant Escherichia coli Nissle 1917 via Human Epidermal Growth Factor Receptor in Human Intestinal Epithelial Cells: Therapeutic Implication Using Recombinant Probiotics

Choi

Ahn²,

Park

et al. 2012

Infect Immun

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The gastrointestinal mucosa has a remarkable ability to repair damage with the support of epidermal growth factor (EGF), which stimulates epithelial migration and proliferative reepithelialization. For the treatment of mucosal injuries, it is important to develop efficient methods for the localized delivery of mucoactive biotherapeutics. The basic idea in the present study came from the assumption that an intestinal probiotic vehicle can carry and deliver key recombinant medicinal proteins to the injured epithelial target in patients with intestinal ulcerative diseases, including inflammatory bowel disease. The study was focused on the use of the safe probiotic E. coli Nissle 1917, which was constructed to secrete human EGF in conjunction with the lipase ABC transporter recognition domain (LARD). Using the in vitro physically wounded monolayer model, ABC transporter-mediated EGF secretion by probiotic E. coli Nissle 1917 was demonstrated to enhance the wound-healing migration of human enterocytes. Moreover, the epithelial wound closure was dependent on EGF receptor-linked activation, which exclusively involved the subsequent signaling pathway of the mitogen-activated protein kinase kinase (MEK) extracellular-related kinases 1 and 2 (ERK1/2). In particular, the migrating frontier of the wounded edge displayed the strongest EGF receptor-linked signaling activation in the presence of the recombinant probiotic. The present study provides a basis for the clinical application of human recombinant biotherapeutics via an efficient, safe probiotic vehicle.

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Section: Methodsmentioning

confidence: 99%

Section: Expression Of Human Egf In E Coli Nissle 1917mentioning

confidence: 99%

Enhanced Wound Healing by Recombinant Escherichia coli Nissle 1917 via Human Epidermal Growth Factor Receptor in Human Intestinal Epithelial Cells: Therapeutic Implication Using Recombinant Probiotics

Choi

Ahn²,

Park

et al. 2012

Infect Immun

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confidence: 99%

“…In P. fluorescens, the ABC protein, MFP, and OMP are encoded by tliD, tliE, and tliF, which are located upstream of tliA in the thermostable lipase operon. The secretion/chaperone domain in the C-terminal region of TliA has been defined as the lipase ABC transporter recognition domain (LARD) (15). Five different fragments of the LARD differing in their lengths have been functionally compared, and LARD3, which contains all four repeats-in-toxin (RTX) motifs, has been identified as the most efficient C-terminal signal for secretion through the ABC transporter (16).…”

mentioning

confidence: 99%

“…In particular, P. fluorescens has a type I secretion system that transports thermostable lipase, TliA, through the ATP-binding cassette (ABC) transporter, TliDEF (14). Its ability to transport several different recombinant proteins has been verified, and the secretion signal has been well researched for its ability to secrete recombinant proteins (15,16).ABC transporters are composed of three subunits: ABC proteins, membrane fusion proteins (MFPs), and outer membrane proteins (OMPs) (17). ABC proteins selectively recognize the secretion domains in the C-terminal regions of the target proteins and hydrolyze ATP to secrete them (18,19).…”

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confidence: 99%

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

Ryu

Lee

Park

et al. 2015

Appl Environ Microbiol

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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...

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Secretion of recombinant proteins from E. coli

Kleiner-Grote

Risse

Friehs

2018

Engineering in Life Sciences

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The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one‐ or two‐step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often‐overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.

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Export of recombinant proteins in Escherichia coli using ABC transporter with an attached lipase ABC transporter recognition domain (LARD)

Cited by 23 publications

References 50 publications

Enhanced Wound Healing by Recombinant Escherichia coli Nissle 1917 via Human Epidermal Growth Factor Receptor in Human Intestinal Epithelial Cells: Therapeutic Implication Using Recombinant Probiotics

Enhanced Wound Healing by Recombinant Escherichia coli Nissle 1917 via Human Epidermal Growth Factor Receptor in Human Intestinal Epithelial Cells: Therapeutic Implication Using Recombinant Probiotics

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

Secretion of recombinant proteins from E. coli

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