Expression and secretion of recombinant ZZ–EGFP fusion protein by the methylotrophic yeast Pichia pastoris.

Jin-bao Tang; Peng Zhu; Hong-ming Yang; Li-min Sun; Shu-liang Song; Ai-guo Ji

We constructed a fusion protein ZZ–EGFP by fusing the ZZ domains of staphylococcal protein A (SpA) and enhanced green fluorescent protein (EGFP). ZZ–EGFP was secreted in the yeast, Pichia pastoris, with a hexahistidine tag. Its expression level was determined by measuring the fluorescence of EGFP. When the recombinant yeast cells in shake-flasks were induced with 0.5% methanol for 96 h, a maximum yield of 115 mg ZZ–EGFP/l was obtained. The resulting ZZ–EGFP fusion protein retained immunoglobulin G (IgG)-binding capacity and EGFP fluorescence. ZZ–EGFP was then used in immunofluorescence assays for detecting antinuclear antibodies (ANA); it produced a good signal that was comparable in its brightness and fluorescence pattern to that generated with fluorescein isothiocyanate (FITC)-labelled anti-human IgG. Thus, ZZ–EGFP showed great potential in immunological applications due to its ability to bind to various IgG from different animal sources.

PICHIA pastoris; STAPHYLOCOCCAL protein A; GREEN fluorescent protein; FLUORESCENCE; YEAST; ANTINUCLEAR factors; IMMUNOGLOBULIN G; IMMUNOLOGY; BIOTECHNOLOGY

Biotechnology Letters, 2008, Vol 30, Issue 8, p1409

0141-5492

Academic Journal

10.1007/s10529-008-9714-5