Browsing by Author "McDade, Kyle Harrison"

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    Developing an in cellulo Carrier-Driven Crystallization System using Cry1Ac from Bacillus thuringiensis
    (2019-09-20) McDade, Kyle Harrison; Fraser, Marie Elizabeth; Turner, Raymond Joseph; Hynes, Michael F.
    X-ray crystallography is the dominant technique for determining the structures of proteins but suffers from two major issues, the crystallization bottleneck and the phase problem. To address these problems, a solution was proposed in which the Cry1Ac protein, which crystallizes spontaneously in cellulo upon sporulation of Bacillus thuringiensis, is used as a crystallization-carrier – to enable the crystallization of auxiliary proteins. Production of endogenous Cry1Ac was optimized through the characterization of sporulation-inducing media: in comparison to C2 and nutrient sporulation media, G-Tris induced the largest inclusions. Cry1Ac was purified using hexane and ultracentrifugation. Non-mechanical lysis methods were developed to aid purification, including the application of B. thuringiensis cell wall lyase, CwlC. Three fusions of cry1ac with the gene of a reporter, green fluorescent protein (gfp) from Aequorea victoria, were created and integrated within Escherichia coli and B. thuringiensis expression systems. Fusions were designed with GFP at the amino-terminus, at the carboxy-terminus and substituting for the toxin domains of Cry1Ac. Production of the fusion proteins within E. coli generated polarizing and fluorescent inclusions that persisted after sonication. Protein fusions and unfused-Cry1Ac produced recombinantly in E. coli failed to generate bipyramidal inclusions characteristic of Cry1Ac. The carboxy-terminal Cry1Ac-GFP fusion and Cry1Ac exhibited similar solubilities when exposed to high pH and chaotropic agents. Recombinant production of protein fusions and unfused-Cry1Ac within the acrystalloferous B. thuringiensis system was poor: Cells rarely contained inclusions, but inclusions of protein fusions appeared bipyramidal and fluorescent. It is suspected that a reduced rate of sporulation caused by the loss of plasmids pHT8, pAW63, and pHT73 resulted in reduced expression. This issue prohibited inclusion purification and further characterization.