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. W...

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Bibliographic Details
Published in:Biotechnology letters 2008-08, Vol.30 (8), p.1409-1414
Main Authors: Tang, Jin-bao, Zhu, Peng, Yang, Hong-ming, Sun, Li-min, Song, Shu-liang, Ji, Ai-guo
Format: Article
Language:English
Subjects:
Applied Microbiology
Biochemistry
Biological and medical sciences
Biomedical and Life Sciences
Biotechnology
Cell Line
Electrophoresis, Polyacrylamide Gel
Fluorescence
Fluorescent Antibody Technique
Fundamental and applied biological sciences. Psychology
Gene expression
Genetic Vectors - genetics
Green Fluorescent Proteins - isolation & purification
Green Fluorescent Proteins - secretion
Humans
Immunoglobulin G - metabolism
Immunoglobulins
Life Sciences
Microbiology
Original Research Paper
Pichia - metabolism
Pichia pastoris
Plasmids - genetics
Protein Structure, Tertiary
Proteins
Recombinant Fusion Proteins - isolation & purification
Recombinant Fusion Proteins - secretion
Staphylococcal Protein A - chemistry
Yeast
Yeasts
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Summary: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.
ISSN:0141-5492
1573-6776
DOI:10.1007/s10529-008-9714-5