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Infect Immun, February 1998, p. 862-865, Vol. 66, No. 2
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Expression and Purification of the Recombinant Lethal Factor of Bacillus anthracis

Pankaj Gupta,1 Smriti Batra,1 Arun P. Chopra,2 Yogendra Singh,2 and Rakesh Bhatnagar1,*

Centre for Biotechnology, Jawahar Lal Nehru University, New Delhi 110067,1 and Centre for Biochemical Technology, Delhi 110007,2 India

Received 9 September 1997/Returned for modification 24 October 1997/Accepted 15 November 1997

The structural gene for the 90-kDa lethal factor (LF) isolated from Bacillus anthracis was expressed as a fusion protein with six histidine residues in Escherichia coli. Expression of LF in E. coli under the transcriptional regulation of the T5 promoter yielded a soluble cytosolic protein with an apparent molecular mass of 90 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant LF reacted with anti-LF antibodies. The protein was purified to homogeneity by nickel nitrilotriacetic acid affinity chromatography and gel filtration on a Sephacryl S-200 column followed by anion exchange on a fast-performance liquid chromatograph with a Resource-Q column. The yield of purified LF from this procedure was 1.5 mg/liter. In solution, trypsin cleaved protective antigen bound to native and recombinant LF with comparable affinities. In macrophage lysis assays, native and recombinant LF exhibited identical potencies. The results suggest that large amounts of biologically active LF can be purified by this procedure.

* Corresponding author. Mailing address: Centre for Biotechnology, Jawahar Lal Nehru University, New Delhi 110067, India. Phone (91) 11-6179751. Fax (91) 11-6865886. E-mail: rakesh{at}jnuniv.ernet.in.




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