Characterization of camel nanobodies specific for superfolder GFP fusion proteins

Mol Biol Rep. 2014 Oct;41(10):6887-98. doi: 10.1007/s11033-014-3575-x. Epub 2014 Aug 2.

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.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibody Specificity / immunology
  • Antigens / immunology
  • Camelus / immunology*
  • Cell Surface Display Techniques
  • Epitope Mapping
  • Female
  • Gene Library
  • Green Fluorescent Proteins / immunology*
  • Immunization
  • Immunoglobulin G / immunology
  • Molecular Sequence Data
  • Peptide Library
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / immunology*
  • Sequence Alignment
  • Single-Domain Antibodies / chemistry
  • Single-Domain Antibodies / genetics
  • Single-Domain Antibodies / immunology*

Substances

  • Antigens
  • Immunoglobulin G
  • Peptide Library
  • Recombinant Fusion Proteins
  • Single-Domain Antibodies
  • Green Fluorescent Proteins