title: Production, characterization, and immunogenicity of a secreted form of Plasmodium falciparum merozoite surface protein 4 produced in Bacillus subtilis.
abstract: Plasmodium falciparum is the causative agent of the most serious form of malaria. Although a combination of control measures has significantly limited malaria morbidity and mortality in the last few years, it is generally agreed that sustained control or even eradication will require additional tools including an effective malaria vaccine. Merozoite surface protein 4, MSP4, which is present during the asexual stage of P. falciparum, is a recognized target that would be useful in a subunit vaccine against blood stages of malaria. Falciparum malaria is most prevalent in developing countries, and this in turn leads to a requirement for safe, low-cost vaccines. We have attempted to utilize the nonpathogenic, gram-positive organism Bacillus subtilis to produce PfMSP4. PfMSP4 was secreted into the culture medium at a yield of 4.5 mg/L. Characterization studies including SDS-PAGE, mass spectrometry, and N-terminal sequencing indicated that the B. subtilis expression system secreted a full length PfMSP4 protein compared to a truncated version in Escherichia coli. Equivalent amounts of purified B. subtilis and E. coli-derived PfMSP4 were used for immunization studies, resulting in statistically significant higher mean titer values for the B. subtilis-derived immunogen. The mouse antibodies raised against B. subtilis produced PfMSP4 that were reactive to parasite proteins as evidenced by immunoblotting on parasite lysate and indirect immunofluorescence assays of fixed parasites. The B. subtilis expression system, in contrast to E. coli, expresses higher amounts of full length PfMSP4 products, decreased levels of aggregates, and allows the development of simplified downstream processing procedures.
authors.affiliation: Department of Chemical engineering, IIT Bombay, Mumbai, 400076, India.
authors.forename: G
authors.initials: G
authors.lastname: Chittibabu
authors.forename: Charles
authors.initials: C
authors.lastname: Ma
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Netter
authors.forename: Santosh B
authors.initials: SB
authors.lastname: Noronha
authors.forename: Ross L
authors.initials: RL
authors.lastname: Coppel
mesh_terms: D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood / Q000276:immunology; D000953:Antigens, Protozoan / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D001412:Bacillus subtilis / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D017780:Malaria Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D013058:Mass Spectrometry; D051379:Mice; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D020539:Sequence Analysis, Protein; D022223:Vaccines, Subunit / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D017780:Malaria Vaccines; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D022223:Vaccines, Subunit; C118839:merozoite surface protein 4, Plasmodium