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Infection and Immunity, October 2001, p. 6532-6536, Vol. 69, No. 10
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.10.6532-6536.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Praveen Kumar, Nidhi Ahuja, and Rakesh Bhatnagar*

Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India

Received 25 April 2001/Returned for modification 11 June 2001/Accepted 9 July 2001

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.

* Corresponding author. Mailing address: Centre for Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India. Phone: (91) 11-6179751. Fax: (91) 11-6198234 or -6865886. E-mail: rakbhat{at}hotmail.com.

Infection and Immunity, October 2001, p. 6532-6536, Vol. 69, No. 10
0019-9567/01/$04.00+0   DOI: 10.1128/IAI.69.10.6532-6536.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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