Modification of an expression vector for efficient recombinant production and purification of mitogillin of Aspergillus fumigatus expressed in Escherichia coli.
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.
Al-Sheikh
Ali
A
Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait.
Ahmad
Suhail
S
Khan
Zia U
ZU
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
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; C036196:MITF protein, Aspergillus restrictus; D012260:Ribonucleases