title: Optimization of the cell wall microenvironment allows increased production of recombinant Bacillus anthracis protective antigen from B. subtilis.
abstract: 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.
authors.affiliation: School of Biochemistry and Genetics, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH.
authors.forename: Joanne E
authors.initials: JE
authors.lastname: Thwaite
authors.forename: Les W J
authors.initials: LW
authors.lastname: Baillie
authors.forename: Noel M
authors.initials: NM
authors.lastname: Carter
authors.forename: Keith
authors.initials: K
authors.lastname: Stephenson
authors.forename: Mark
authors.initials: M
authors.lastname: Rees
authors.forename: Colin R
authors.initials: CR
authors.lastname: Harwood
authors.forename: Peter T
authors.initials: PT
authors.lastname: Emmerson
mesh_terms: D000409:Alanine / Q000378:metabolism; D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D001408:Bacillus anthracis / Q000235:genetics* / Q000276:immunology; D001412:Bacillus subtilis / Q000235:genetics* / Q000254:growth & development / Q000276:immunology / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000737:chemistry* / Q000502:physiology; D017077:Culture Media, Conditioned; D009154:Mutation; D009876:Operon; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D017077:Culture Media, Conditioned; D011994:Recombinant Proteins; D000409:Alanine