PROTEIN EXPRESSION AND PURIFICATION 7, 33–38 (1996) Article No. 0005 Expression and Purification of Anthrax Toxin Protective Antigen from Escherichia coli Manju Sharma, Prabodha K. Swain, Arun P. Chopra, Vijay K. Chaudhary,* and Yogendra Singh Genetic Engineering Division, Centre for Biochemical Technology, Mall Road, Delhi 110 007, India; and *Biochemistry Department, South Campus, University of Delhi, New Delhi 110 021, India Received February 28, 1995, and in revised form August 8, 1995 LF is believed to be a zinc-dependent metalloprotease Anthrax toxin consists of three separate proteins, (7). Lethal toxin has been found to cause an early influx protective antigen (PA), lethal factor (LF), and edema of sodium and efflux of potassium from J774A.1 cells, factor (EF). PA binds to the receptor on mammalian leading to ATP depletion resulting in lysis of cells (8). cells and facilitates translocation of EF or LF into the PA alone is nontoxic, an essential immunogen, and the cytosol. PA is the primary component of several an- primary component of the human vaccine against an- thrax vaccines. In this study we expressed and purified thrax (9,10). PA from Escherichia coli. The purification of PA from The PA gene has been cloned, sequenced (11), and E. coli was possible after transporting the protein into expressed in B. subtilis (12), Escherichia coli (13), and the periplasmic space using the outer membrane pro- viruses (14). The purification of PA from sonicates of tein A signal sequence. The purification involved se- E. coli was not successful due to low expression and quential chromatography through hydroxyapatite, extensive degradation by E. coli cellular proteases (15). DEAE Sepharose CL-4B, followed by Sephadex G-100. In this study attempts have been made to transport The typical yield of purified PA from this procedure the PA protein into the periplasmic space to reduce was 500 mg/liter. PA expressed and purified from E. degradation. Most proteins translocated across bacte- coli was similar to the PA purified from Bacillus an- rial membranes are synthesized as protein precursors, thracis in its ability to lyse a macrophage cell line containing a 15–20 amino acid extension at the N- (J774A.1). The present results suggest that a signal se- terminus called signal peptide (16). Apart from the sig- quence is required for the efficient translocation of PA nal peptide, the ultimate localization of the proteins into E. coli periplasmic space.  1996 Academic Press, Inc. among the inner membrane, periplasm, or outer mem- brane depends on structural information contained within the protein itself (17). In this paper, we report that the addition of the sig- The virulence of Bacillus anthracis, the causative nal sequence for the outer membrane protein A (OmpA) agent of anthrax, depends on two factors, a poly-D-glu- of E. coli at the 5-end of the PA gene results in the tamic acid capsule and a three component protein exo- efficient transport of the expressed PA protein to the toxin. The genes coding for the toxin and the enzymes periplasmic space. The accumulation of PA in the per- responsible for capsule production are carried on B. iplasm facilitated protein purification by protecting PA anthracis plasmids pXOI and pXO2, respectively (1,2). from degradation by isolating it from several cellular The three proteins of the exotoxins are protective anti- proteases. The deletion of 14 amino acids at the COOH- gen (PA), lethal factor (LF), and edema factor (EF). terminus resulted in the formation of inclusion bodies. These proteins are individually nontoxic, but in combi- Addition of the OmpA signal sequence could not enable nation form two distinct toxins causing different patho- the transport of the COOH-terminus deleted PA into genic responses in animals and cultured cells (3,4). Pro- the periplasm. tective antigen binds to a cell surface receptor and facil- itates the internalization of EF or LF and their MATERIALS AND METHODS translocation into the cytosol (5,6). EF is a calmodulin- dependent adenylate cyclase which increases cAMP to Reagents. Restriction enzymes and chemicals were purchased from New England BioLabs (Beverley, MA) nonphysiological concentrations in eukaryotic cells (5). 33 1046-5928/96 $12.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.