title: Expression of Histophilus somni IbpA DR2 protective antigen in the diatom Thalassiosira pseudonana.
abstract: Increasing demand for the low-cost production of valuable proteins has stimulated development of novel expression systems. Many challenges faced by existing technology may be overcome by using unicellular microalgae as an expression platform due to their ability to be cultivated rapidly, inexpensively, and in large scale. Diatoms are a particularly productive type of unicellular algae showing promise as production organisms. Here, we report the development of an expression system in the diatom Thalassiosira pseudonana by expressing the protective IbpA DR2 antigen from Histophilus somni for the production of a vaccine against bovine respiratory disease. The utilization of diatoms with their typically silicified cell walls permitted development of silicon-responsive transcription elements to induce protein expression. Specifically, we demonstrate that transcription elements from the silicon transporter gene SIT1 are sufficient to drive high levels of IbpA DR2 expression during silicon limitation and growth arrest. These culture conditions eliminate the flux of cellular resources into cell division processes, yet do not limit protein expression. In addition to improving protein expression levels by molecular manipulations, yield was dramatically increased through cultivation enhancement including elevated light and CO2supplementation. We substantially increased recombinant protein production over starting levels to 1.2% of the total sodium dodecyl sulfate-extractable protein in T. pseudonana, which was sufficient to conduct preliminary immunization trials in mice. Mice exposed to 5 μg of diatom-expressed DR2 in whole or sonicated cells (without protein purification) exhibited a modest immune response without the addition of adjuvant.
authors.affiliation: Marine Biology Research Division, Scripps Institution of Oceanography, 9500 Gilman Dr, La Jolla, CA, 92093, USA.
authors.forename: Aubrey
authors.initials: A
authors.lastname: Davis
authors.affiliation: Department of Pathology, School of Medicine, UC San Diego, San Diego, CA, USA.
authors.forename: Lauren T
authors.initials: LT
authors.lastname: Crum
authors.affiliation: Department of Pathology, School of Medicine, UC San Diego, San Diego, CA, USA.
authors.forename: Lynette B
authors.initials: LB
authors.lastname: Corbeil
authors.affiliation: Marine Biology Research Division, Scripps Institution of Oceanography, 9500 Gilman Dr, La Jolla, CA, 92093, USA. mhildebrand@ucsd.edu.
authors.forename: Mark
authors.initials: M
authors.lastname: Hildebrand
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D001428:Bacterial Vaccines / Q000235:genetics / Q000276:immunology; D002245:Carbon Dioxide / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D002418:Cattle Diseases / Q000382:microbiology / Q000517:prevention & control*; D017377:Diatoms / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D008027:Light; D051379:Mice; D015661:Pasteurellaceae / Q000235:genetics*; D016871:Pasteurellaceae Infections / Q000276:immunology / Q000517:prevention & control / Q000662:veterinary*; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D012141:Respiratory Tract Infections / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control / Q000662:veterinary; D012825:Silicon / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001428:Bacterial Vaccines; D011994:Recombinant Proteins; D014157:Transcription Factors; D014614:Vaccines, Synthetic; D002245:Carbon Dioxide; D012825:Silicon
keywords: Diatom; Histophilus somni; IbpA DR2; Microalgae; Protective antigen; Thalassiosira pseudonana; Valuable proteins