title: Characterization of camel nanobodies specific for superfolder GFP fusion proteins.
abstract: Superfolder green fluorescent protein (sfGFP) is a fusion tag which plays a dual role in monitoring and purifying the recombinant fusion proteins using specific binders. Nanobodies are the smallest intact antigen binding fragments derived from heavy chain-only antibodies (HCAbs) occurring in camelids. They are produced as recombinant proteins in E. coli and have different biotechnological applications, including the detection and purification of their specific antigens. To produce anti-sfGFP specific nanobodies, an adult one-humped camel was successfully immunized and immune response was evaluated by ELISA, which showed an active participation of HCAbs in this response. A relatively large nanobody "immune" library of 5 × 10(8) individual transformants, with 87.5 % positivity, was prepared from the blood of the immunized camel. Phage display biopanning on this nanobody library resulted in the isolation of seven anti-sfGFP specific nanobodies, referred to as NbsfGFP01, 02, 03, 04, 06, 07 and 08. These nanobodies were able to recognize sfGFP tag as free or in fusion with growth hormone in ELISA and immuno-blotting. Furthermore, they showed important apparent affinities in the detection and capture of sfGFP by ELISA, and they targeted three different epitopes on the surface of their antigen. The interesting characteristics of these molecular binders make them valuable tools for more in-depth structural and functional studies related to sfGFP fusion proteins.
authors.affiliation: Department of Biotechnology, Faculty of Agriculture, Damascus University, Damascus, Syria.
authors.forename: Aya
authors.initials: A
authors.lastname: Twair
authors.forename: Souad
authors.initials: S
authors.lastname: Al-Okla
authors.forename: Moutaz
authors.initials: M
authors.lastname: Zarkawi
authors.forename: Abdul Qader
authors.initials: AQ
authors.lastname: Abbady
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity / Q000276:immunology; D000941:Antigens / Q000276:immunology; D002162:Camelus / Q000276:immunology*; D061505:Cell Surface Display Techniques; D018604:Epitope Mapping; D005260:Female; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000276:immunology*; D007114:Immunization; D007074:Immunoglobulin G / Q000276:immunology; D008969:Molecular Sequence Data; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology*; D016415:Sequence Alignment; D061905:Single-Domain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
chemical_list: D000941:Antigens; D007074:Immunoglobulin G; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; D049452:Green Fluorescent Proteins
references.citation: Immunol Lett. 2009 Apr 27;123(2):103-13
references.pmid: 19428557
references.citation: J Mol Biol. 2007 Oct 19;373(2):476-90
references.pmid: 17822714
references.citation: Methods Mol Biol. 2012;911:15-26
references.pmid: 22886243
references.citation: Vet Immunol Immunopathol. 2009 Mar 15;128(1-3):178-83
references.pmid: 19026455
references.citation: J Chromatogr B Analyt Technol Biomed Life Sci. 2008 Mar 15;864(1-2):116-22
references.pmid: 18313994
references.citation: J Immunol Methods. 2008 Jan 1;329(1-2):138-50
references.pmid: 17996887
references.citation: Mol Cell Proteomics. 2008 Feb;7(2):282-9
references.pmid: 17951627
references.citation: Antimicrob Agents Chemother. 2001 Oct;45(10):2807-12
references.pmid: 11557473
references.citation: Trends Biochem Sci. 2001 Apr;26(4):230-5
references.pmid: 11295555
references.citation: Blood. 1997 Nov 1;90(9):3316-21
references.pmid: 9345013
references.citation: Mol Microbiol. 2002 Sep;45(5):1191-6
references.pmid: 12207688
references.citation: PLoS One. 2008 Jun 11;3(6):e2351
references.pmid: 18545653
references.citation: Gene. 1992 Feb 15;111(2):229-33
references.pmid: 1347277
references.citation: Methods Mol Biol. 1996;58:491-510
references.pmid: 8713899
references.citation: Appl Microbiol Biotechnol. 2007 Nov;77(1):13-22
references.pmid: 17704915
references.citation: Nat Biotechnol. 2005 Jan;23(1):102-7
references.pmid: 15580262
references.citation: Nature. 1993 Jun 3;363(6428):446-8
references.pmid: 8502296
references.citation: EMBO J. 2000 Mar 1;19(5):921-30
references.pmid: 10698934
references.citation: Nat Biotechnol. 2006 Jan;24(1):79-88
references.pmid: 16369541
references.citation: J Cell Comp Physiol. 1962 Jun;59:223-39
references.pmid: 13911999
references.citation: Protein Expr Purif. 2006 Jul;48(1):1-13
references.pmid: 16427311
references.citation: Nat Biotechnol. 1996 Mar;14(3):315-9
references.pmid: 9630892
references.citation: Gene. 2014 May 25;542(1):69-76
references.pmid: 24534464
references.citation: Nat Methods. 2006 Oct;3(10):845-54
references.pmid: 16990817
references.citation: J Mol Biol. 2008 Mar 21;377(2):478-88
references.pmid: 18262543
references.citation: J Mol Recognit. 1999 Mar-Apr;12(2):131-40
references.pmid: 10398404
references.citation: Biochim Biophys Acta. 2003 Dec 5;1624(1-3):21-8
references.pmid: 14642809
references.citation: Nat Methods. 2012 Jun;9(6):582-4
references.pmid: 22543348
references.citation: Protein Sci. 2010 Dec;19(12):2389-401
references.pmid: 20945358
references.citation: J Biomed Biotechnol. 2009;2009:591923
references.pmid: 20182554
references.citation: Vet Immunol Immunopathol. 2011 Jul 15;142(1-2):49-56
references.pmid: 21592585
references.citation: Nanomedicine (Lond). 2013 Jun;8(6):1013-26
references.pmid: 23730699
references.citation: Nat Struct Mol Biol. 2011 Dec 11;19(1):117-21
references.pmid: 22157958
references.citation: J Biotechnol. 2010 Jan 15;145(2):93-8
references.pmid: 19861136
references.citation: Anal Chem. 2006 May 1;78(9):3072-9
references.pmid: 16642995
references.citation: Nat Biotechnol. 2005 Sep;23(9):1126-36
references.pmid: 16151406
references.citation: Biotechnol Genet Eng Rev. 2007;24:1-30
references.pmid: 18059626
references.citation: Int J Parasitol. 2009 Apr;39(5):625-33
references.pmid: 19041315
references.citation: Protein Sci. 2002 Mar;11(3):500-15
references.pmid: 11847273
references.citation: Nat Struct Mol Biol. 2010 Jan;17(1):133-8
references.pmid: 20010839
references.citation: Arch Biochem Biophys. 2012 Oct 15;526(2):114-23
references.pmid: 22583807
references.citation: Curr Opin Pharmacol. 2008 Oct;8(5):600-8
references.pmid: 18691671
references.citation: FEBS Lett. 1997 Sep 15;414(3):521-6
references.pmid: 9323027