Expression and characterization of Cryptococcus neoformans recombinant App1

Mycopathologia. 2012 Jun;173(5-6):395-405. doi: 10.1007/s11046-011-9486-7. Epub 2011 Oct 5.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carrier Proteins / biosynthesis*
  • Carrier Proteins / chemistry*
  • Carrier Proteins / genetics
  • Cell Line
  • Chromatography, Gel
  • Circular Dichroism
  • Disulfides
  • Electrophoresis, Polyacrylamide Gel
  • Gene Expression
  • Genetic Vectors
  • Insecta
  • Lentivirus / genetics
  • Molecular Conformation
  • Molecular Weight
  • Plasmids
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Spectrometry, Fluorescence

Substances

  • App1 protein, Cryptococcus neoformans
  • Carrier Proteins
  • Disulfides
  • Recombinant Proteins