Enhancing the secretory yields of leech carboxypeptidase inhibitor in Escherichia coli: Influence of trigger factor and signal recognition particle

Puertas, Juan Miguel; Nannenga, Brent L.; Dornfeld, Kevin T.; Betton, Jean‐Michel; Baneyx, François

doi:10.1016/j.pep.2010.06.008

Cited by 14 publications

(20 citation statements)

References 35 publications

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“…We sought to take advantage of this phenotype to identify E. coli mutants that were more efficient at functional MP expression. In initial screen design experiments, TF-deficient (Δ tig ) cells [20] harboring plasmid pHtdR200, a ColE1 derivative encoding a hexahistidine-tagged version of the htdR gene under transcriptional control of the P BAD promoter [5], were plated onto LB agar plates supplemented with L -arabinose and all- trans retinal and incubated for 36 h at 37°C. While most colonies were small and a light shade of red due to the toxicity of HtdR overexpression, we isolated a spontaneous mutant that was both large and purple.…”

Section: Resultsmentioning

confidence: 99%

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Enhanced expression of membrane proteins in E. coli with a PBAD promoter mutant: synergies with chaperone pathway engineering strategies

Nannenga

Baneyx

2011

Microb Cell Fact

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BackgroundMembrane proteins (MPs) populate 20-30% of genomes sequenced to date and hold potential as therapeutic targets as well as for practical applications in bionanotechnology. However, MP toxicity and low yields in normally robust expression hosts such as E. coli has curtailed progress in our understanding of their structure and function.ResultsUsing the seven transmembrane segments H. turkmenica deltarhodopsin (HtdR) as a reporter, we isolated a spontaneous mutant in the arabinose-inducible PBAD promoter leading to improved cell growth and a twofold increase in the recovery of active HtdR at 37°C. A single transversion in a conserved region of the cyclic AMP receptor protein binding site caused the phenotype by reducing htdR transcript levels by 65%. When the mutant promoter was used in conjunction with a host lacking the molecular chaperone Trigger Factor (Δtig cells), toxicity was further suppressed and the amount of correctly folded HtdR was 4-fold that present in the membranes of control cells. More importantly, while improved growth barely compensated for the reduction in transcription rates when another polytopic membrane protein (N. pharonis sensory rhodopsin II) was expressed under control of the mutant promoter in wild type cells, a 4-fold increase in productivity could be achieved in a Δtig host.ConclusionsOur system, which combines a downregulated version of the tightly repressed PBAD promoter with a TF-deficient host may prove a valuable alternative to T7-based expression for the production of membrane proteins that have so far remained elusive targets.

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Section: Resultsmentioning

confidence: 99%

“…E. coli BW25113 [Δ (araD-araB)567 Δ lacZ4787 (::rrnB-3) λ - rph-1 D (rhaD-rhaB)568, hsdR514 ] [26], KTD101 [BW25113 Δ tig100 ] [20], and plasmid pHtdR200 [5] have been described previously. KTD101(pHtdR200) cells were grown to A 600 ≈ 0.45 in 125 mL flasks containing 25 mL of LB media supplemented with 50 μg/mL kanamycin.…”

Section: Methodsmentioning

confidence: 99%

Enhanced expression of membrane proteins in E. coli with a PBAD promoter mutant: synergies with chaperone pathway engineering strategies

Nannenga

Baneyx

2011

Microb Cell Fact

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“…These six expression vectors were transformed in the strain KTD101, which is defective for the expression of the ribosomal protein trigger factor (TF). In a related study performed with LCI as a model protein [17], it was described how deletion of TF resulted in higher levels of secreted proteins via the SRP pathway.…”

Section: Methodsmentioning

confidence: 99%

Expression of metallocarboxypeptidase inhibitors in Escherichia coli: effect of cysteine content and protein size in the secretory production of disulfide-bridged proteins

Puertas

Caminal

González

2011

J Ind Microbiol Biotechnol

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Metallocarboxypeptidase inhibitors are proteins with possible applications in biomedicine given their properties as anticoagulant and antitumoral factors. They are small, eukaryotic polypeptides comprising several disulfide bridges, which makes them hard to express in inexpensive bacterial hosts. In this work, three of them were produced in high-cell-density cultures of Escherichia coli: PCI (39 residues and three bridges), LCI (66 residues and four bridges) and TCI (75 residues and six bridges). The genes coding for the mentioned inhibitors were cloned in an arabinose-inducible plasmid fused to the signal peptide of DsbA in order to have them secreted and grant the formation of the bridges. The trigger-factor defective strain KTD101 was used as the expression host. The resulting recombinant strains were cultured in fed-batch mode employing minimal media and an exponential feed profile, keeping the specific growth rate at μ = 0.1 h(-1) by limitation of the fed carbon source (glycerol). Between 380 and 540 mg l(-1) of active inhibitors were obtained in both the periplasmic extracts and extracellular media of the cultures. Later on, excretion was enhanced using a cell permeabilization treatment, allowing the recovery of over 80% of the products from the extracellular fraction. Protein yields were found to be inversely proportional to cysteine content of the inhibitor, whereas protein excretion rates were inversely proportional to the protein size. Overall, these results offer insight into the secretory production of active disulfide-bridged proteins in high-cell-density cultures of E. coli.

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“…The SRP pathway shares the SecYEG translocon with the Sec pathway (Beckwith, ), but the cytoplasmic aggregation can be effectively prevented since the translocation coincidentally occurs during the protein synthesis (co‐translational translocation) (Puertas et al, ). Thereby, the SRP pathway has been especially more useful for the production of easy‐to‐aggregate proteins such as the fast‐folding proteins originated from unrelated organisms (Lee et al, ; Puertas et al, ; Steiner et al, ) or inner membrane proteins that have multiple transmembrane segments (Nannenga and Baneyx, ; Skretas et al, ). However, severe growth defects were reported occasionally when excess proteins were secreted via the SRP pathway (Lee et al, ; Makino et al, ).…”

Section: Introductionmentioning

confidence: 99%

“…In order to solve this problem, several efforts such as controlling the endogenous level of SRP pathway‐related biomolecules have been made. Until now, the co‐expression of Ffh (protein component of SRP), 4.5S RNA (encoded by ffs , RNA component of SRP), or FtsY (SRP receptor) is known to be effective for SRP‐dependent protein production (Lee et al, ; Nannenga and Baneyx, ; Puertas et al, ). In addition, it was discovered that knocking out Trigger Factor (TF, encoded by tig ) could improve the SRP machinery, promoting the competitive binding of SRP to the ribosome (Nannenga and Baneyx, ; Puertas et al, ).…”

Section: Introductionmentioning

confidence: 99%

Enhanced secretion of recombinant proteins via signal recognition particle (SRP)‐dependent secretion pathway by deletion of rrsE in Escherichia coli

Lee

et al. 2016

Biotech & Bioengineering

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Although signal recognition particle (SRP)-dependent secretion pathway, which is characterized by co-translational translocation, helps prevent cytoplasmic aggregation of proteins before secretion, its limited capacity for the protein secretion is a major hurdle for utilizing the pathway as an attractive route for secretory production of recombinant proteins. Therefore, we developed an Escherichia coli mutant, whose efficiency of secretion via the SRP pathway was dramatically increased. First, we developed a novel FACS-based screening system by combining a periplasmic display system (PECS) and direct fluorescent labeling with the organoarsenic compound, FlAsH-EDT2 . With this screening system, transposon-insertion library of E. coli was screened, and then we isolated mutants which exhibited higher protein production through the SRP pathway than the parental strain. From the genetic analysis, we found that all isolated mutants had the same mutation-disruption of the 16S rRNA gene (rrsE). The positive effect of rrsE deficiency on protein secretion via the SRP pathway was successfully demonstrated using various model proteins including endogenous SRP-dependent proteins, antibodies, and G protein-coupled receptor. For the large-scale production of IgG and GPCR, we performed fed-batch cultivation with the rrsE-deficient mutant, and very high yields of IgG (0.4 g/L) and GPCR (1.4 g/L) were obtained. Biotechnol. Bioeng. 2016;113: 2453-2461. © 2016 Wiley Periodicals, Inc.

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Enhancing the secretory yields of leech carboxypeptidase inhibitor in Escherichia coli: Influence of trigger factor and signal recognition particle

Cited by 14 publications

References 35 publications

Enhanced expression of membrane proteins in E. coli with a PBAD promoter mutant: synergies with chaperone pathway engineering strategies

Enhanced expression of membrane proteins in E. coli with a PBAD promoter mutant: synergies with chaperone pathway engineering strategies

Expression of metallocarboxypeptidase inhibitors in Escherichia coli: effect of cysteine content and protein size in the secretory production of disulfide-bridged proteins

Enhanced secretion of recombinant proteins via signal recognition particle (SRP)‐dependent secretion pathway by deletion of rrsE in Escherichia coli

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