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Applied and Environmental Microbiology, January 2002, p. 227-234, Vol. 68, No. 1
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.1.227-234.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Optimization of the Cell Wall Microenvironment Allows Increased Production of Recombinant Bacillus anthracis Protective Antigen from B. subtilis

Joanne E. Thwaite,1,2,{dagger} Les W. J. Baillie,2 Noel M. Carter,3,{ddagger} Keith Stephenson,3,§ Mark Rees,3,¶ Colin R. Harwood,3 and Peter T. Emmerson1*

School of Biochemistry and Genetics,1 Defence Science and Technical Laboratory, Chemical and Biological Sciences, Porton Down, Salisbury, Wiltshire SP4 0JQ, United Kingdom,2 Department of Microbiology and Immunology, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH3

Received 11 July 2001/ Accepted 8 October 2001

The stability of heterologous proteins secreted by gram-positive bacteria is greatly influenced by the microenvironment on the trans side of the cytoplasmic membrane, and secreted heterologous proteins are susceptible to rapid degradation by host cell proteases. In Bacillus subtilis, degradation occurs either as the proteins emerge from the presecretory translocase and prior to folding into their native conformation or after the native conformation has been reached. The former process generally involves membrane- and/or cell wall-bound proteases, while the latter involves proteases that are released into the culture medium. The identification and manipulation of factors that influence the folding of heterologous proteins has the potential to improve the yield of secreted heterologous proteins. Recombinant anthrax protective antigen (rPA) has been used as a model secreted heterologous protein because it is sensitive to proteolytic degradation both before and after folding into its native conformation. This paper describes the influence of the microenvironment on the trans side of the cytoplasmic membrane on the stability of rPA. Specifically, we have determined the influence of net cell wall charge and its modulation by the extent to which the anionic polymer teichoic acid is D-alanylated on the secretion and stability of rPA. The potential role of the dlt operon, responsible for D-alanylation, was investigated using a Bacillus subtilis strain encoding an inducible dlt operon. We show that, in the absence of D-alanylation, the yield of secreted rPA is increased 2.5-fold. The function of D-alanylation and the use of rPA as a model protein are evaluated with respect to the optimization of B. subtilis for the secretion of heterologous proteins.

* Corresponding author. Mailing address: School of Biochemistry and Genetics, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom. Phone: 44 (0) 191 222 7434. Fax: 44 (0) 191 222 7424. E-mail: P.T.Emmerson{at}ncl.ac.uk.

{dagger} Present address: Dstl, Chemical and Biological Sciences, Porton Down, Salisbury, Wiltshire SP4 0JQ, United Kingdom.

{ddagger} Present address: Dept. of Surgery, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, United Kingdom.

§ Present address: Dept. of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037.

Present address: Nova Castra Laboratories Ltd., Newcastle upon Tyne NE12 3EW, United Kingdom.

Applied and Environmental Microbiology, January 2002, p. 227-234, Vol. 68, No. 1
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.1.227-234.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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