Expression and purification of ZEBRA fusion proteins and applications for the delivery of macromolecules into mammalian cells.
The recent development of peptide carriers for efficient and specific delivery of biologically active molecules into mammalian cells represents a major advance in the study of both normal and uncontrolled cell growth. In the past few years, this technology has been successfully applied to the delivery of therapeutic molecules in animal models, and now some of these carriers are available in the clinic for the treatment of some human diseases. This unit describes the production, in a bacterial expression system, of reporter proteins (EGFP and beta-galactosidase) fused to a transduction domain of the Epstein-Barr virus ZEBRA protein, as well as purification of the fusion proteins. The purified fusion proteins can be added to any of a large spectrum of mammalian cells and the internalization process measured by flow cytometry and fluorescence microscopy on live cells. Fluorescence microscopy on fixed cells is used to study their intracellular distribution.
Rothe
Romy
R
HumProTher Laboratory, TheREx-GREPI, University of Joseph Fourier, UFR de Médecine, La Tronche, France.
Lenormand
Jean-Luc
JL
HumProTher Laboratory, TheREx-GREPI, University of Joseph Fourier, UFR de Médecine, La Tronche, France.
D000818:Animals; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000737:chemistry / Q000235:genetics; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification*; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
D011993:Recombinant Fusion Proteins; D054334:Viral Regulatory and Accessory Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase