Graduate Thesis Or Dissertation
 

Creation of an efficient protein expression and secretion system in the gram-positive bacterial vector, Streptococcus gordonii

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  • Streptococcus gordonii (S. gordonii) is being developed as a Gram-positive expression vector for the expression of heterologous proteins. This bacterial expression system takes advantage of the natural pathway of protein export and anchoring on the surface of the bacterium. The expression of heterologous proteins relies on the homologous recombination of a plasmid into the genome of S. gordonii. The heterologous proteins are expressed constitutively in the bacterium and exported. In order to purify the proteins away from the bacterium; the surface anchoring motifs have been removed from the expressed protein. In this way, the proteins are directly secreted into the medium. Results show that the protein is no longer associated with the bacterium, but is present in the supernatant. In addition, about 3 mg/l of protein can be produced without any optimization of growth conditions. In order to purify these proteins away from the contaminating medium components, a histidine tag was added to either the N or the C-terminus of the secreted heterologous protein. These constructs were tested for their ability to bind to a metal affinity column and elute under native conditions. The results of these experiments show that the proteins do bind and elute under the appropriate conditions and that the proteins can be purified 10-15 fold from the medium. The addition of an affinity tag can be problematic depending on the type of protein expressed. Perhaps enzyme activity is affected or the protein is intended for therapeutic use. In this case, the affinity tag would need to be removed from the target protein. In order to both purify a protein and remove the purification tag, secreted fusions were constructed with a target protein fused to an intein and chitin binding domain. These proteins were tested for the ability to bind chitin beads and cleave between the target protein and the intein. Results show that binding and cleavage do occur under the appropriate conditions. These data demonstrate that S. gordonii can be used to express and secrete a variety of functional proteins that can be purified to allow their downstream use as research tools, therapeutics, or vaccine antigens.
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