Selection of a fully human single domain antibody specific to Helicobacter pylori urease

Fouladi, Mehdi; Sarhadi, Shamim; Tohidkia, Mohammadreza; Fahimi, Farnaz; Samadi, Nasser; Sadeghi, Javid; Barar, Jaleh; Omidi, Yadollah

doi:10.1007/s00253-019-09674-6

Cited by 19 publications

(18 citation statements)

References 47 publications

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“…Then, the overnight culture was diluted in 1:100 ratio by 350 mL of fresh 2xYT-0.1% Glc-Amp medium in a 2 L culture media bottle and incubated at 37 °C until reached an optical density at 600 nm (OD 600 ) of 0.9. The induction of anti-G17-Gly scFv expression was performed with IPTG at a final concentration of 1 mM at 30 °C for 5 h through the experiment, unless otherwise specified (Fahimi et al 2018 ; Fouladi et al 2019 ).…”

Section: Materials Methodsmentioning

confidence: 99%

Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies

et al. 2020

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Single-chain variable fragments (scFvs) have gained increased attention among researchers in both academic and industrial fields owing to simple production in E. coli. The E. coli periplasm has been the site of choice for the expression of scFv molecules due to its oxidizing milieu facilitating correctly formation of disulfide bonds. Hence, the recovery of high-yield and biologically active species from the periplasmic space is a critical step at beginning of downstream processing. TES (Tris/EDTA/Sucrose) as a simple and efficient extraction method has been frequently used but under varied extraction conditions, over literature. This study, for the first time, aimed to interrogate the effects of four independent variables (i.e., Tris–HCl concentration, buffer’s pH, EDTA concentration, and incubation time) and their potential interactions on the functional extraction yield of an scFv antibody from the periplasmic space of E. coli . The results indicated that the Tris–HCl concentration and pH are the most significant variables in the TES method and displayed a positive effect at their lower values on the functional extraction yield. Besides, the statistical analysis revealed 4 significant interactions between different variables. Here is the first report on the successful application of a design of experiment based on a central composite design to establish a generic and optimal TES extraction condition. Accordingly, an optimal condition for TES extraction of scFv molecules from the periplasm of HB2151 at the exponential phase was developed as follows: 50 mM Tris–HCl at pH 7.2, 0.53 mM EDTA, and an incubation time of 60 min.

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Section: Materials Methodsmentioning

confidence: 99%

Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies

et al. 2020

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“…Human single-fold semisynthetic phage-scFv library (Tomlinson I + J), E. coli strains: TG1 (T-phage resistant) and HB2151, and KM13 helper phage were obtained from Source BioScience (Nottingham Business Park, Nottingham, UK). 21,22 The library was constructed by the insertion of scFv-encoding genes approximate to gIII in a phagemid vector containing ampicillin resistance marker (pIT2) and transformed into TG1 strain. The amber stop codon located between scFv and gIII sequences allows for either the display of scFv-pIII fusion proteins in the suppressor strain TG1 or production of free soluble scFvs in the non-suppressor strain HB2151.…”

Section: Methodsmentioning

confidence: 99%

“…27 The next day, 2 µL of this culture was inoculated to a new plate containing 200 µL/well of 2xTY-Amp-Glc and grown until the OD 600 reached 0.4. 21 After infection with KM13 helper phage, the culture medium was altered to 2xTY-Amp-Glc-Kan (supplemented with 100 µg/mL ampicillin, 0.1% glucose, and 50 µg/mL kanamycin) and incubated shaking overnight at 30°C. The scFv-displaying phages rescued into the supernatants of the bacterial cultures were subsequently utilized for ELISA as described above.…”

Section: Methodsmentioning

confidence: 99%

Successful Application of Whole Cell Panning for Isolation of PhageAntibody Fragments Specific to Differentiated Gastric Cancer Cells

et al. 2019

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Purpose: Generation of antibodies which potentially discriminate between malignant and healthy cells is an important prerequisite for early diagnosis and treatment of gastric cancer (GC). Comparative analysis of cell surface protein landscape will provide an experimental basis for biomarker discovery, which is essential for targeted molecular therapies. This study aimed to isolate phage-displayed antibody fragments recognizing cell surface proteins, which were differently expressed between two closely related GC cell lines, namely AGS and MKN-45. Methods: We selected and screened a semisynthetic phage-scFv library on AGS, MKN-45, and NIH-3T3 cell lines by utilizing a tailored selection scheme that was designed to isolate phagescFvs that not only recognize the differentiated AGS cells but also distinguish them from NIH3T3 fibroblasts and the poorly differentiated MKN-45 cells. Results: After four rounds of subtractive whole cell panning, 14 unique clones were identified by ELISA screening and nucleotide sequencing. For further characterization, we focused on four phage-scFvs with strong signals in screening, and their specificity was confirmed by cell-based ELISA. Furthermore, the selected phage-scFvs were able to specifically stain AGS cells with 38.74% (H1), 11.04% (D11), 76.93% (G11), and 69.03% (D1) in flow cytometry analysis which supported the ability of these phage scFvs in distinguishing AGS from MKN-45 and NIH-3T3 cells. Conclusion: Combined with other proteomic techniques, these phage-scFvs can be applied to membrane proteome analysis and, subsequently, identification of novel tumor-related antigens mediating proliferation and differentiation of cells. Furthermore, such antibody fragments can be exploited for diagnostic purposes as well as targeted drug delivery of GC.

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“…1 Biopanning steps (modified from Kügler et al). (1) mimicked binding phages to targets, (2) washing away unbound phages, (3) eluting off specific bound phages, (4) enriching the specifically bound phages for next round of biopanning In vitro biopanning is usually chosen for a variety of targets on cell surface or molecules, which includes biopanning on the immobilized molecules (Rahbarnia et al 2017 ; Zhao et al 2018 ), solution biopanning (Fouladi et al 2019 ; Thanongsaksrikul et al 2018 ), selection on whole cells (Carneiro et al 2014 ; Li et al 2017 ; Yeh et al 2016 ), reverse biopanning (Ferraro et al 2013 ), display of established internalizing peptides (Rangel et al 2012 ). Molecules screening by this approach are more likely to keep native conformation, and recognize specific post-translational modifications (Kehoe et al 2006 ; Velappan et al 2019 ).…”

Section: Biopanningmentioning

confidence: 99%

Application of Phage-Displayed Peptides in Tumor Imaging Diagnosis and Targeting Therapy

Xü

et al. 2020

Int J Pept Res Ther

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Phage display is an effective and powerful technique that provides a route to discovery unique peptides targeting to tumor cells. Specifically binding peptides are considered as the valuable target directing molecule fragments with potential efficiency to improve the current tumor clinic, and offer new approaches for tumor prevention, diagnosis and treatment. We focus on the recent advances in the isolation of tumor-targeting peptides by biopanning methods, with particular emphasis on molecular imaging, and pharmaceutical targeting therapy.

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Selection of a fully human single domain antibody specific to Helicobacter pylori urease

Cited by 19 publications

References 47 publications

Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies

Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies

Successful Application of Whole Cell Panning for Isolation of PhageAntibody Fragments Specific to Differentiated Gastric Cancer Cells

Application of Phage-Displayed Peptides in Tumor Imaging Diagnosis and Targeting Therapy

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Selection of a fully human single domain antibody specific to Helicobacter pylori urease

Cited by 19 publications

References 47 publications

Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies

Optimization of Tris/EDTA/Sucrose (TES) periplasmic extraction for the recovery of functional scFv antibodies

­­­Successful Application of Whole Cell Panning for Isolation of PhageAntibody Fragments Specific to Differentiated Gastric Cancer Cells

Application of Phage-Displayed Peptides in Tumor Imaging Diagnosis and Targeting Therapy

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Successful Application of Whole Cell Panning for Isolation of PhageAntibody Fragments Specific to Differentiated Gastric Cancer Cells