Construction of chimeric proteins between protein G and fluorescence-enhanced green fluorescent protein, and their application to immunoassays

Chimeric proteins consisting of an IgG-binding domain of protein G (PG) and a fluorescence-enhanced green fluorescent protein variant (EGFP) were constructed to evaluate their utility in fluoroimmunoassays (FIAs). Four chimeric proteins (PG-EGFPs) with an amino acid residue of Ala, Pro, Ser or Thr as the linker between the two domains were constructed and expressed in E. coli. They were efficiently expressed and could be purified to homogeneity with one-step IgG-affinity chromatography. The proteins retained similar fluorescence spectra to those of EGFP, and the binding constants, as evaluated using BIAcore, revealed their high affinity for rabbit IgG, goat IgG, sheep IgG and mouse IgG(1). PG-EGFP with Ala, Pro, Ser as the linker showed a high affinity for rabbit IgG while that with Thr had a 3-fold lower binding constant. When PG-EGFPs were applied to sandwich FIAs, a minimum detection limit of 0.1 mu g/ml of BSA using anti-BSA antibody, or 1 mu g/ml of rabbit IgG was achieved using microplate format. This detection sensitivity was comparable to that obtained using FITC-labeled secondary antibodies. Western blotting of hen egg lysozyme and BSA using PG-EGFPs was also possible with similar sensitivity. These data show the applicability of these easily prepared PG-EGFPs to various FIAs for sensitive detection of antigens.