Publication Database

The baculovirus expression system is suitable to purify palmitoylated proteins - functional studies with the t-SNARE SNAP-25 (2003)

Art

Vortrag

Autoren

Kammer, B.

Schmidt, M. F. G.

Veit, M.

Kongress

International Congress on Protein Expression and Protein Function

Berlin, 25. – 28.05.2003

Quelle

International Congress on Protein Expression and Protein Function, Berlin

— S. 63

Sprache

Englisch

Kontakt

Institut für Immunologie

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 51834
immunologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Palmitoylation is the covalent attachment of fatty acids to cysteine residues of membrane proteins. The fatty acids are thought to effect protein-protein interactions and/or anchor hydrophilic proteins to membranes. Functional studies with purified palmitoylated proteins have rarely been performed, because recombinant proteins expressed in E. coli lack their authentic modifications and purification of a palmitoylated protein from its natural environement is usually difficult.
SNARE-proteins are key players in neurosecretion. Interactions between v-SNAREs located on synaptic vesicles and t-SNAREs on the presynaptic membrane drive membrane fusion. After fusion NSF and a-SNAP disassemble the SNARE-complex into its individual components. In contrast to the other SNAREs, the t-SNARE SNAP-25 does not contain a hydrophobic transmembrane region, but is palmitoylated at four cysteines in the middle of the molecule.
To study the role of palmitoylation in-vitro, we have expressed 6xHis-SNAP-25 with recombinant baculovirus in insect cells. Ni-affinity chromatography is sufficient to obtain an apparently pure protein. Metabolic labeling with [3H]-palmitate and Triton-X-114 phase distribution proved that SNAP-25 is palmitoylated in significant amounts. Palmitoylated as well as non-acylated SNAP-25 form SNARE-complexes which could then be disassembled by NSF and a-SNAP. Palmitoylated SNAP-25 attaches almost quantitatively to liposomes, whereas only small amounts of non-acylated SNAP-25 bind to these artificial membranes. Thus, palmitoylation of SNAP-25 is not required for the protein-protein interactions, but for stable membrane anchoring of this intrinsically hydrophilic protein. The results also show that the baculovirus expression system is suitable to purify palmitoylated proteins for functional studies.