Eukaryotic expression and secretion of EGFP-labeled annexin A5.
The Ca2+-dependent binding of annexin A5 to phosphatidylserine on cell surfaces is a reliable marker for apoptosis that is widely used in flow cytometry based apoptosis assays. In this approach, annexin A5 must be coupled to a fluorescent dye, but standard dyes such as fluorescein are photolabile, and the heterogeneous chemical linkage partially inhibits binding to phosphatidylserine. Recombinant fusions comprising annexin A5 and fluorescent proteins are available for prokaryotic expression, but can be purified only at low concentrations due to their low solubility in the cytoplasm. Here we describe a eukaryotic expression system for the secretion of functional recombinant annexin A5, with and without fluorescent protein fusions, in different formats. Metal affinity purification yielded up to 18 microg of histidine-tagged annexin A5 fusions per ml processed cell culture supernatants. Furthermore the supernatant itself was sufficient for direct use in apoptosis assays. The availability of such fusion proteins offers new and more economical opportunities for the development and application of this widely utilized apoptosis assay.
Stöcker
Michael
M
Institute for Molecular Biotechnology, Forckenbeckstr. 6, 52074 Aachen, Germany. stoecker@molbiotech.rwth-aachen.de
Pardo
Alessa
A
Hetzel
Christian
C
Reutelingsperger
Chris
C
Fischer
Rainer
R
Barth
Stefan
S
D017304:Annexin A5 / Q000096:biosynthesis* / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006801:Humans; D007668:Kidney; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D020298:U937 Cells
D017304:Annexin A5; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins