Graduate Thesis Or Dissertation
 

Advances in expression of heterologous protein products by Streptococcus gordonii

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  • Streptococcus gordonii is a bacterial species that naturally colonizes the oral cavity of most healthy humans. It resides in the mouth as an adherent to dental surfaces and, with few exceptions, does not cause disease in individuals it inhabits. It possesses qualities that encourage its use as a vector to deliver human vaccines against pathogens that enter at mucosal surfaces such as the mouth, throat, lungs, gastrointestinal or urogenital tracts. S. gordonii is easily engineered in the laboratory to produce foreign, or heterologous, proteins derived from pathogenic organisms. Thus, S. gordonii engineered to produce particular viral may stimulate protective immunity against smallpox, a potential biowarfare agent, without causing the harmful side effects associated with the current vaccine. The research presented in this dissertation desribes new ways that S. gordonii can be genetically engineered for greater production of heterologous products derived from pathogens. S. gordonii naturally secretes two proteins, glucosyltransferase and amylase-binding protein during growth, and the genetics S. gordonii utilizes to produce these products were reproduced and situated on a plasmid to facilitate production of a heterologous product. Although some plasmids constructs could not be introduced into E. coli, it was found that the addition of a synthetic peptide to growth medium enhanced the efficiency with which S. gordonii assimilated genetic material, thereby allowing the plasmid to be directly introduced into S. gordonii. The expression plasmid is well retained by S. gordonii that colonized the oral cavities of mice. The mechanisms that S. gordonii uses to export products from the cell are capable of accommodating high-level expression of at least two heterologous products, smallpox vaccine candidates A27L and B5R. Further, particular viral products that had previously proven recalcitrant to expression were produced by S. gordonii by engineering the fusion of viral sequences with those derived from a bacterial protein.
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file details Dissertation_Compilation.pdf 2017-08-18 Pubblico Scaricare