Multifunctional g3p-peptide tag for current phage display systems

Beckmann, Christiane; Haase, Bernd; Timmis, Kenneth N.; Tesar, Michael

doi:10.1016/s0022-1759(98)00008-8

Cited by 13 publications

(6 citation statements)

References 18 publications

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“…The supernatants from induced cultures of E. coli HB2151 (pVDL9.3) cells grown at 37°C and harbouring p6AC3HLYA or pEHLYA were used as the source of 6AC3HLYA and EHLYA polypeptides respectively. The samples of scFv 6AC3 and an unrelated scFv (named B4) used as a control (Beckman et al ., 1998) were purified from the periplasm of E. coli HB2151 harbouring p6AC3g3 or pB4g3 respectively. The protein concentrations of scFvs and HlyA derivatives used in these assays were serial threefold dilutions ranging from 0.5 to 0.01 µg ml −1 .…”

Section: Resultsmentioning

confidence: 99%

Formation of disulphide bonds during secretion of proteins through the periplasmic‐independent type I pathway

Fernández

Lorenzo²

2001

Molecular Microbiology

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SummaryIn this work, we have investigated whether the bacterial type I secretion pathway, which does not have a periplasmic intermediate of the secreted protein, allows the formation of disulphide bridges. To this end, the formation of disulphide bonds has been studied in an antibody single-chain Fv (scFv) fragment secreted by the Escherichia coli haemolysin (Hly) transporter (a paradigm of type I secretion). The scFv antibody fragment was used as a disulphide bond and protein-folding reporter, as it contains two disulphide bridges that are required for its correct folding (i.e. to preserve its antigen-binding activity). We show that an scFv±HlyA hybrid secreted by Hly type I transporter (TolC, HlyB, HlyD) is accumulated in the extracellular medium with the disulphide bonds correctly formed. Neither periplasmic and inner membrane-bound Dsb enzymes (e.g. DsbC, DsbG, DsbB and DsbD) nor cytoplasmic thioredoxins (TrxA and TrxC) were required for scFv±HlyA oxidation. However, a mutation of the thioredoxin reductase gene (trxB), which leads to the cytoplasmic accumulation of the oxidized forms of thioredoxins, had a specific inhibitory effect on the Hly-dependent secretion of disulphide-containing proteins. These data suggest that premature cytoplasmic oxidation of the substrate may interfere with the secretion process. Taken together, these results indicate not only that the type I system tolerates secretion of disulphidecontaining proteins, but also that disulphide bonds are specifically formed during the passage of the polypeptide through the export conduit.

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Section: Resultsmentioning

confidence: 99%

Formation of disulphide bonds during secretion of proteins through the periplasmic‐independent type I pathway

Fernández

Lorenzo²

2001

Molecular Microbiology

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“…In order to demonstrate that Nluc can be used as a reporter system and its small size (171 amino acids) neither compromises efficient bacterial production nor the function of a fusion partner, a scFv format has been chosen in a first application. For that purpose a Nluc expression vector was constructed which allowed the C-terminal fusion of single scFv genes or scFv pools to the N-terminus of the Nluc reporter gene via a 24 aa linker sequence derived from Promega vector pNL3.1 ( Figure 2A ) For direct comparison and calibration of hit rates the detection via anti-pIII ( Tesar et al, 1995 ; Beckmann et al, 1998 ; Mersmann et al, 1998 ) and anti-Strep-tag ® II ( Knappik and Brundiers, 2009 ) was used. Five distinct immune phage libraries generated in-house were subjected to three rounds of a standard phage panning on immobilized protein ( Hoogenboom et al, 1998 ) and analyzed for specific scFvs directly expressed from the phagemid vector pPL101 via anti-pIII or, after subcloning into the two different expression vectors pPL302 via Nluc or pPL303 ( Figure 2A ), via an anti-Strep Tag II ( Knappik and Brundiers, 2009 ).…”

Section: Resultsmentioning

confidence: 99%

NanoLuc Luciferase – A Multifunctional Tool for High Throughput Antibody Screening

et al. 2016

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Based on the recent development of NanoLuc luciferase (Nluc), a small (19 kDa), highly stable, ATP independent, bioluminescent protein, an extremely robust and ultra high sensitivity screening system has been developed whereby primary hits of therapeutic antibodies and antibody fragments could be characterized and quantified without purification. This system is very versatile allowing cellular and solid phase ELISA but also homogeneous BRET based screening assays, relative affinity determinations with competition ELISA and direct Western blotting. The new Nluc protein fusion represents a “swiss army knife solution” for today and future high throughput antibody drug screenings.

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“…Peptide arrays were prepared on amino-PEG500 cellulose membrane-UC540 (Intavis AG, Cologne, Germany) using a SPOT robot (Intavis AG) according to a standard spot-synthesis protocol 26. Special cellulose chromatography paper was chemically derived to carry spots of dipeptide anchors for the preparation of either immobilized peptide.…”

Section: Methodsmentioning

confidence: 99%

<p>Establishing a detection method for CCNY: a potentially significant clinical investigative marker in NSCLC patients</p>

Teng

et al. 2019

OTT

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BackgroundCCNY, a novel cyclin family member, plays an increasingly important role in the progression of tumor invasion and metastasis, including lung cancer. However, the clinical significance of CCNY in non-small-cell lung cancer (NSCLC) patients is unknown.Patients and methodsWe prepared CCNY monoclonal antibodies, validated specific peptides by a peptide array, and established a double-antibody sandwich ELISA detection method. Then, we measured CCNY levels in 100 NSCLC patients and 50 healthy controls. A blinded validation was subsequently performed in 399 NSCLC patients and 150 healthy controls.ResultsWe successfully prepared two specific mouse anti-human CCNY monoclonal antibodies and established a reliable and stable detection method. In the training set, serum CCNY was markedly increased in the NSCLC patients (P<0.05) with an integrated area under the curve of 0.751. With further analysis of the CCNY levels, there were no differences in age, sex, smoking status, tumor location, histologic subtype, or tumor size, but differences were observed in lymphatic (P<0.001) and distant (P<0.001) metastases in NSCLC patients. The CCNY[+] patients had a shorter survival time and progression-free survival than CCNY[−] patients at 3-year follow-up (P<0.001). The results were confirmed by the validation set.ConclusionOur study suggests that CCNY may be useful as a latent tumor marker to facilitate diagnosis and may be an effective indicator of tumor aggressiveness, playing an important role in the prognosis of NSCLC patients.

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Multifunctional g3p-peptide tag for current phage display systems

Cited by 13 publications

References 18 publications

Formation of disulphide bonds during secretion of proteins through the periplasmic‐independent type I pathway

Formation of disulphide bonds during secretion of proteins through the periplasmic‐independent type I pathway

NanoLuc Luciferase – A Multifunctional Tool for High Throughput Antibody Screening

<p>Establishing a detection method for CCNY: a potentially significant clinical investigative marker in NSCLC patients</p>

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