NDLI: Purification of Anthrax Edema Factor from Escherichia coli and Identification of Residues Required for Binding to Anthrax Protective Antigen

Content Provider	PubMed Central
Author	Kumar, Praveen Ahuja, Nidhi Bhatnagar, Rakesh
Editor	Barbieri, J. T.
Copyright Year	2001
Abstract	The structural gene for anthrax edema factor (EF) was expressed in Escherichia coli under the control of a powerful T5 promoter to yield the 89-kDa recombinant protein that reacted with anti-EF antibodies. Recombinant EF was purified to homogeneity by a two-step procedure involving metal chelate affinity chromatography and cation-exchange chromatography. From 1 liter of culture, 2.5 mg of biologically active EF was easily purified. This is the first report of purification of anthrax EF from E. coli. EF purified from E. coli was biologically and functionally as active as its Bacillus anthracis counterpart. The recombinant protein could compete with lethal factor for binding to protective antigen. Sequence analysis revealed a stretch of seven amino acids, Val Tyr Tyr Glu Ile Gly Lys, present both in EF (residues 136 to 142) and lethal factor (residues 147 to 153). To investigate the role of these seven residues in binding to protective antigen, the residues were individually mutated to alanine in EF. Mutations in residues Tyr137, Tyr138, Ile140, and Lys142 of EF specifically blocked its interaction with anthrax protective antigen. The adenylate cyclase activity of the mutants remained unaffected. The results suggested that residues Tyr137, Tyr138, Ile140, and Lys142 are required for binding of EF to anthrax protective antigen, which facilitates its entry into susceptible cells.
Related Links	http://dx.doi.org/10.1128/iai.69.10.6532-6536.2001
Ending Page	6536
Page Count	5
Starting Page	6532
File Format	PDF
ISSN	00199567
e-ISSN	10985522
Journal	Infection and Immunity
Issue Number	10
Volume Number	69
Language	English
Publisher	American Society for Microbiology
Publisher Date	2001-10-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Immunology Microbiology Parasitology Infectious Diseases Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Infectious Diseases Parasitology Immunology Microbiology

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