Expression and Characterization of Cryptococcus neoformans Recombinant App1
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We characterized Cryptococcus neoformans recombinant antiphagocytic protein 1 (rApp1) by SDS–PAGE, gel filtration chromatography, circular dichroism, and fluorescence spectroscopy. rApp1 produced by C. neoformans var. grubii contains an odd number of cysteines; therefore, it has the ability to form intermolecular disulfide bridges which can lead to the formation of amyloid fibrils in the absence of β-mercaptoethanol or DTT in vitro. Alternate approaches to over-expression of rApp1 in the Lepidopteran High Five™ Insect cell line using pIZ/V5-His and in lentivirus were explored and are described. Finally, localization of App1 in vivo was examined in the presence and absence of the capsule.
KeywordsCryptococcus neoformans Antiphagocytic protein 1 (App1) Intermolecular disulfide bridges Insect cells Lentivirus
We are grateful to Dr Brendan Duggan for very helpful discussions and Drs Mark Kindy and Salim El-Amouri from the Department of Neuroscience at MUSC for their help with lentivirus expression. We also thank Rick Peppler from the MUSC Flow Cytometry & Cell Sorting (FCCS) Shared Resource Facility for carrying out the flow cytometry work. This work was supported in part by the Burroughs Wellcome Fund, the National Institutes of Health (grants AI56168, AI71142 and AI87541 to M. D. P.), and the Centers of Biomedical Research Excellence Program of the National Center for Research Resources (grant RR17677 project 2 to M. D. P.). Dr Maurizio Del Poeta is a Burroughs Wellcome New Investigator in the Pathogenesis of Infectious Diseases.