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

• To increase solubility and immunogenicity, GH was produced as fusion with sfGFP.
• A camel was immunized with GFP-GH and a large nanobody (Nb) library was constructed.
• Five different anti-GH specific Nbs were retrieved by phage display and characterized.
• Anti-GH specific Nbs are valuable tools for productive and diagnostic purposes.

Growth hormone (GH) deficiencies are diagnosed in most children with short stature and treated with a long course of administrating expensive and daily doses of recombinant human GH (rhGH or Somatropin®). This work describes for the first time the production of several GH specific nanobodies with great potential in the field of GH production and detection. Nanobodies are the smallest intact antigen binders derived from heavy chain-only antibodies (HCAbs) of camelids. They are very stable, highly soluble and are produced as recombinant proteins in Escherichiacoli at an affordable cost for various biotechnological applications.To increase its solubility and immunogenicity, GH was produced as fusion with superfolder green fluorescent protein (sfGFP) and was used in this form to successfully immunize an adult camel. The active involvement of HCAbs in the specific camel immune response encouraged the preparation of large nanobody “immune” library. Phage display biopanning of this library against GH resulted in the isolation of five interesting and different nanobodies, referred to as NbGH01, 02, 03, 04 and 06. All nanobodies were able to recognize GH in its fusion and free formats and the detection sensitivity ranged from 0.5 to 10 ng/ml in sandwich ELISA. Pure rhGH was successfully purified by affinity chromatography, using immobilized NbGH06, from the cleavage reaction of fusion proteins with the tobaccos etch virus (TEV) protease. These specific molecular binders, especially NbGH06, provide valuable tools for rhGH diagnostic as well as for production purposes.

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