Production ofChlamydia pneumoniaeProteins inBacillus subtilisand Their Use in Characterizing Immune Responses in the Experimental Infection Model

Abstract

Due to intracellular growth requirements, large-scale cultures of chlamydiae and purification of its proteins are difficult and laborious. To overcome these problems we produced chlamydial proteins in a heterologous host, Bacillus subtilis, a gram-positive nonpathogenic bacterium. The genes of Chlamydia pneumoniae major outer membrane protein (MOMP), the cysteine-rich outer membrane protein (Omp2), and the heat shock protein (Hsp60) were amplified by PCR, and the PCR products were cloned into expression vectors containing a promoter, a ribosome binding site, and a truncated signal sequence of the ␣-amylase gene from Bacillus amyloliquefaciens. C. pneumoniae genes were readily expressed in B. subtilis under the control of the ␣-amylase promoter. The recombinant proteins MOMP and Hsp60 were purified from the bacterial lysate with the aid of the carboxy-terminal histidine hexamer tag by affinity chromatography. The Omp2 was separated as an insoluble fraction after 8 M urea treatment. The purified proteins were successfully used as immunogens and as antigens in serological assays and in a lymphoproliferation test. The Omp2 and Hsp60 antigens were readily recognized by the antibodies appearing after pulmonary infection following intranasal inoculation of C. pneumoniae in mice. Also, splenocytes collected from mice immunized with MOMP or Hsp60 proteins proliferated in response to in vitro stimulation with the corresponding proteins.

Dual role of infections as risk factors for coronary heart disease

“…The blood vials were put on ice and IgG and IgA antibodies against the C. pneumoniae HSP60 were measured with EIA as described in detail elsewhere [7]. Briefly, microtiter plates were coated with a recombinant C. pneumoniae HSP60 protein produced in Bacillus subtilis [8] at a concentration of 5 µg/ml in PBS (pH 7.4) overnight at 37°C. After coating, the plates were incubated for 2 h at 37°C with duplicate samples were grown in appropriate cell cultures.…”

Bacillus subtilis as a tool for vaccine development: from antigen factories to delivery vectors

“…In both cases the reduced immunogenicity of the antigens expressed by the B. subtilis strains was attributed to the lack of conformational epitopes, lost during expression or purification steps, required for induction of protective antibody responses. Three Chlamydia pneumoniae proteins, two outer membrane proteins and one cytoplasmic heatshock inducible protein, were cloned in expression plasmid vectors and produced in B. subtilis strains either as insoluble (membrane proteins) or soluble cytoplasmic proteins with histidine tags (Airaksinen et al 2003). Purified proteins elicited specific serum antibodies, after parenteral administration to mice, which reacted with the native proteins expressed by C. pneumonia strains and induced cell proliferation after exposure of splenocytes to elementary bodies (Airaksinen et al 2003).…”

“…Three Chlamydia pneumoniae proteins, two outer membrane proteins and one cytoplasmic heatshock inducible protein, were cloned in expression plasmid vectors and produced in B. subtilis strains either as insoluble (membrane proteins) or soluble cytoplasmic proteins with histidine tags (Airaksinen et al 2003). Purified proteins elicited specific serum antibodies, after parenteral administration to mice, which reacted with the native proteins expressed by C. pneumonia strains and induced cell proliferation after exposure of splenocytes to elementary bodies (Airaksinen et al 2003). Other reports have also described the use of recombinant B. subtilis strains for the production of pneumolysin, a pneumococcal toxin, and an outer membrane protein of Haemophilus influenzae, either as secreted or cytoplasmic proteins, encoded by multi-copy plasmidbased expression systems (Srikumar et al 1993, Taira et al 1989).…”

Development and Characterization of a Subtilin-Regulated Expression System in Bacillus subtilis : Strict Control of Gene Expression by Addition of Subtilin

“…The starch-inducible amylase promoter is frequently used for production of heterologous proteins in which the desired protein is fused to the ␣-amylase promoter and leader peptide, which efficiently drives secretion of the protein produced into the culture medium (1,17). Several prophagederived heat-inducible gene expression systems that show very tight control of gene expression have been described; however, the levels of expression upon maximum induction are relatively low compared to those of other inducible gene expression systems (7,17,31).…”

“…Bacillus subtilis is generally considered to have great industrial potential for production and secretion of proteins of clinical interest, like interferon (37), insulin (36), pathogenic antigens (1), and toxins (45), or enzymes of great industrial interest, like proteases (17), ␣-amylase (18), and lipases (17). The major advantages of B. subtilis compared to other host production systems are high-cell-density growth and secretion of the synthesized protein into the cultivation medium, which facilitates isolation and purification of the protein during downstream processing (5,31).…”