The aim of this study is to express the receptor-binding domain of Bacillus anthracis protective antigen in E.coli . Signal sequence of the outer membrane protein A (OmpA) of E.coli was attached to the 5′ end of the ...The aim of this study is to express the receptor-binding domain of Bacillus anthracis protective antigen in E.coli . Signal sequence of the outer membrane protein A (OmpA) of E.coli was attached to the 5′ end of the gene encoding protective antigen receptor-binding domain (the 4 th domain of PA, PAD4). The plasmid carrying the fusion gene was then transformed into E.coli and induced to express recombinant PAD4 by IPTG. The recombinant protein was purified by chromatography and then identified by N-terminal sequencing and Western blot. The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ion-exchange chromatography and gel filtration, about 10 mg of homogenous recombinant PAD4 was obtained from 1 L culture. Data from N-terminal sequencing suggested that the amino acid sequence of recombinant PAD4 was identical with its natural counterpart. And the result of Western blot showed the recombinant protein could bind with anti-PA serum from rabbit. High level secreted expression of PAD4 was obtained in E.coli . The results reported here are parts of a continuing research to evaluate PAD4 as a potential drug for anthrax therapy or a candidate of new vaccine.展开更多
文摘The aim of this study is to express the receptor-binding domain of Bacillus anthracis protective antigen in E.coli . Signal sequence of the outer membrane protein A (OmpA) of E.coli was attached to the 5′ end of the gene encoding protective antigen receptor-binding domain (the 4 th domain of PA, PAD4). The plasmid carrying the fusion gene was then transformed into E.coli and induced to express recombinant PAD4 by IPTG. The recombinant protein was purified by chromatography and then identified by N-terminal sequencing and Western blot. The recombinant protein, about 10% of the total bacterial protein in volume, was secreted to the periplasmic space of the cell. After a purification procedure including ion-exchange chromatography and gel filtration, about 10 mg of homogenous recombinant PAD4 was obtained from 1 L culture. Data from N-terminal sequencing suggested that the amino acid sequence of recombinant PAD4 was identical with its natural counterpart. And the result of Western blot showed the recombinant protein could bind with anti-PA serum from rabbit. High level secreted expression of PAD4 was obtained in E.coli . The results reported here are parts of a continuing research to evaluate PAD4 as a potential drug for anthrax therapy or a candidate of new vaccine.
