Expression and secretion of recombinant ZZ-EGFP fusion protein by the methylotrophic yeast Pichia pastoris.
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.
Tang
Jin-Bao
JB
Department of Basic Medicine, Weifang Medical University, WeiFang 261042, People's Republic of China.
Zhu
Peng
P
Yang
Hong-ming
HM
Sun
Li-Min
LM
Song
Shu-liang
SL
Ji
Ai-guo
AG
D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D010843:Pichia / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000737:chemistry*
D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins