title: Modification of an expression vector for efficient recombinant production and purification of mitogillin of Aspergillus fumigatus expressed in Escherichia coli.
abstract: The diagnostic potential of secretory proteins of Aspergillus fumigatus is limited by their availability in pure form. We have constructed a vector (pGES-PH-1) to express genes encoding secretory proteins of A. fumigatus as fusion proteins with glutathione S-transferase (GST) in Escherichia coli. The mitogillin, a secretary protein of A. fumigatus, was expressed and purified to homogeneity by using pGES-PH-1. Mitogillin gene was PCR amplified from A. fumigatus DNA, cloned in pGES-PH-1 and expressed in E. coli as fusion protein with GST at N-terminal and 6xHis tag at C-terminal end. Pure mitogillin was obtained by purification on glutathione-Sepharose, cleavage of column-bound fusion protein by PreScission protease and by further purification on Ni-NTA-agarose. Polyclonal anti-mitogillin antibodies were raised in rabbits and were used to study its secretion during in vitro growth of A. fumigatus. The mitogillin was detectable in culture filtrate after 24 h of A. fumigatus growth and thereafter its amount increased progressively until 96 h in both, Sabouraud dextrose broth and potato dextrose broth. However, the secretion of mitogillin in culture medium was slightly delayed when A. fumigatus was grown in a minimal medium as mitogillin was detected only after 36 h of growth. Our results demonstrate the utility of the newly constructed expression vector with two affinity tags and PreScission protease cleavage site for high-level expression and efficient purification of a recombinant A. fumigatus secretory protein expressed in E. coli, which could be used for further studies.
authors.affiliation: Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait.
authors.forename: Ali
authors.initials: A
authors.lastname: Al-Sheikh
authors.forename: Suhail
authors.initials: S
authors.lastname: Ahmad
authors.forename: Zia U
authors.initials: ZU
authors.lastname: Khan
mesh_terms: D000818:Animals; D001232:Aspergillus fumigatus; D001483:Base Sequence; D002846:Chromatography, Affinity; D004926:Escherichia coli; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D005822:Genetic Vectors; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D012260:Ribonucleases / Q000096:biosynthesis* / Q000302:isolation & purification
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; C036196:MITF protein, Aspergillus restrictus; D012260:Ribonucleases