Characterization of camel nanobodies specific for superfolder GFP fusion proteins Aya Twair • Souad Al-Okla • Moutaz Zarkawi • Abdul Qader Abbady Received: 17 June 2013 / Accepted: 30 June 2014 / Published online: 2 August 2014 Ó Springer Science+Business Media Dordrecht 2014 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 bio- technological 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 9 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. Keywords Camel Á Nanobody Á sfGFP Á Phage display Á Fusion protein List of abbreviations AP Alkaline phosphatase BCIP 5-Bromo-4-chloro-3-indolyl phosphate BSA Bovine serum albumin ELISA Enzyme-linked immunosorbent assay FPLC Fast protein liquid chromatography HCAb Heavy-chain antibody HRP Horseradish peroxidase IPTG Isopropyl b-D-thiogaldctoside NBT Nitro blue tetrazolium chloride PBS Phosphate buffered saline RFLP Restriction fragment length polymorphism scFv Single chain antibody variable fragment SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis TMB 3,3 0 ,5,5 0 -tetramethylbenzidine Introduction The green fluorescent protein (GFP) from Aequorea jelly- fish has become a versatile tag for many in vivo and in vitro applications because of its ability to fold and form A. Twair Department of Biotechnology, Faculty of Agriculture, Damascus University, Damascus, Syria A. Twair Á A. Q. Abbady (&) Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS), Damascus, Syria e-mail: ascientific@aec.org.sy S. Al-Okla Department of Animal Biology, Faculty of Sciences, Damascus University, Damascus, Syria M. Zarkawi Department of Agriculture, AECS, P. O. Box 6091, Damascus, Syria 123 Mol Biol Rep (2014) 41:6887–6898 DOI 10.1007/s11033-014-3575-x