Generation and characterization of nanobodies against rhGH expressed as sfGFP fusion protein

Abbady, Abdul Qader; Al-Shemali, Rasha; Assaad, Jana Mir; Murad, Hossam

doi:10.1016/j.ygcen.2014.05.018

Cited by 9 publications

(16 citation statements)

References 71 publications

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“…However, in this study we choose to add an N-terminal 6× His tag to the different forms of annexin V and use it in the well-defined method of nickel-charged column to achieve the purification. The efficiency of the 6× His tag at the N-terminal of sf GFP in metal affinity chromatography has previously proven in different studies (Al-Homsi et al, 2012a, 2015; Abbady et al, 2014; Twair et al, 2014). The PS-affinity, even though it is laborious and expensive, has one great advantage over nickel affinity chromatography for annexin V purification in that it provides a direct proof of the functionality of the expressed protein (Ernst et al, 1998).…”

Section: Discussionmentioning

confidence: 97%

“…In our previous works, sf GFP was successfully applied as an innovative tag for expressing short peptides (Al-Homsi et al, 2015) and recovering growth hormone (GH) from the inclusion bodies (Abbady et al, 2014). Several anti- sf GFP specific nanobodies, recombinant single chain antibodies from camel, were also prepared and could have interesting applications in GFP-fusion protein technology (Twair et al, 2014).…”

Section: Discussionmentioning

confidence: 99%

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Characterization of Annexin V Fusion with the Superfolder GFP in Liposomes Binding and Apoptosis Detection

et al. 2017

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Programed cell death is a critical and unavoidable part of life. One of the most widely used markers for dying cells, by apoptosis or pyroptosis, is the redistribution of phosphatidylserine (PS) from the inner to the outer plasma membrane leaflet. Annexin V protein is a sensitive and specific probe to mark this event because of its high affinity to the exposed PS. Beyond that, annexin V can bind to any PS-containing phospholipid bilayer of almost all tiny forms of membranous vesicles like blood platelets, exosomes, or even nanostructured liposomes. In this work, recombinant human annexin V was produced as a fusion with a highly fluorescent superfolder derivative of the green fluorescent protein (sfGFP) in Escherichia coli. The fusion protein(sfGFP-ANXV, 64 kDa), annexin V (ANXV, 40 kDa), and sfGFP (27 kDa) were separately produced after cloning their encoding genes in pRSET plasmid, and all proteins were expressed in a soluble form, then purified in high yields because of their N-terminal 6× His tag (~150 mg of pure protein per 1 L culture). Superiority of this fluorescent fusion protein over fluorescein-conjugated annexin V was demonstrated in binding to phospholipids (and their liposomes), prepared from natural sources (soya bean and egg yolk) that have different content of PS, by using different methods including ELISA, dot-blotting, surface plasmon resonance, and flow cytometry. We also applied fluorescent annexin V in the detection of apoptotic cells by flow cytometry and fluorescent microscopy. Interestingly, sfGFP-ANXV fusion was more sensitive to early apoptotic stressed HeLa cells than fluorescein-conjugated-ANXV. This highly expressed and functional sfGFP-ANXV fusion protein provides a promising ready-to-use molecular tool for quantifying liposomes (or similarly exosomes) and detecting apoptosis in cells.

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Characterization of Annexin V Fusion with the Superfolder GFP in Liposomes Binding and Apoptosis Detection

et al. 2017

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“…Nanobodies are procured by cloning their genetic repertoire from B cells circulating in the blood of an immunized camel, constructing a cDNA library and panning by phage display [10,12]. Several published reports described the involvement of HCAbs in camelid immunity, especially in the response to pathogenic antigens [4,13,14].…”

Section: Elisamentioning

confidence: 99%

“…Different antigens of recombinant human growth hormone (rhGH) from previous work [12] were immobilized on nitrocellulose membrane by spotting 2 μl (1 μg) of the samples at the center of the grid, and the membrane was left to dry. Blocking was conducted with 5× blocking buffer in TBS-T, then spots were treated with or without NbGH01 nanobody (1/500) [12] then with different antibodies: R-anti-camel rIgG (1:1000), R-anti6×His (Bethyl Laboratories Inc., 1:5000), R-anti-GFP (homemade, 1:3000) [20], and R-anti-GH (homemade, 1:3000) [21].…”

Section: Dot Blottingmentioning

confidence: 99%

“…Blocking was conducted with 5× blocking buffer in TBS-T, then spots were treated with or without NbGH01 nanobody (1/500) [12] then with different antibodies: R-anti-camel rIgG (1:1000), R-anti6×His (Bethyl Laboratories Inc., 1:5000), R-anti-GFP (homemade, 1:3000) [20], and R-anti-GH (homemade, 1:3000) [21]. After three washes with TBS-T, primary antibodies were detected with secondary conjugated AP goat anti-rabbit (1:3000).…”

Section: Dot Blottingmentioning

confidence: 99%

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Purification of polyclonal IgG specific for Camelid’s antibodies and their recombinant nanobodies

et al. 2016

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Camelid’ s heavy-chain antibody (HCAb) consists of only two heavy chains and lacks the two light chains together with the CH1 domain usually found in conventional immunoglobulins. A recombinant single antigen-binding entity, named VHH (or Nanobody®) was generated by reengineering the variable domains from HCAb. This study focuses on the detection of camelid´s immunoglobulins as well as their derivative nanobodies using a universal anti-camel antibody produced in rabbit (rIgG). Starting from a crude rabbit serum, a standard stock of rIgG (1 mg/ml) was prepared after purification by affinity chromatography using protein-A column. As expected, rIgG was able to detect camel antibodies in ELISA and immunoblotting, and its reactivity was equal against all different camel IgG subclasses, which were purified from serum by differential affinity chromatography on protein-G and -A. Interestingly, rIgG also recognized nanobodies since they were originally part of camel HCAbs, providing an alternative method to detect the corpus of these recombinant proteins rather than targeting their artificial tags. These data suggest that the anti-camel rIgG described here could be efficiently applied at different stages of nanobody technology, including the quantitation of the issued nanobodies and their detection when bound to target antigens.

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Assessing the bioactivity of the codon optimized sfGFP-IGF1 fusion protein via interaction with IGFBP3 and induction of cell proliferation

et al. 2019

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Generation and characterization of nanobodies against rhGH expressed as sfGFP fusion protein

Cited by 9 publications

References 71 publications

Characterization of Annexin V Fusion with the Superfolder GFP in Liposomes Binding and Apoptosis Detection

Characterization of Annexin V Fusion with the Superfolder GFP in Liposomes Binding and Apoptosis Detection

Purification of polyclonal IgG specific for Camelid’s antibodies and their recombinant nanobodies

Assessing the bioactivity of the codon optimized sfGFP-IGF1 fusion protein via interaction with IGFBP3 and induction of cell proliferation

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