Synapsin IIa: expression in insect cells, purification, and characterization.
Synapsin IIa belongs to a family of neuron-specific phosphoproteins called synapsins, which are associated with synaptic vesicles in presynaptic nerve terminals. In order to examine the biochemical properties of synapsin IIa, and ultimately its physiological function, purified protein is required. Since attempts to purify significant quantities of synapsin IIa, an isoform of the synapsins, from mammalian brain have proven difficult, we undertook the production of recombinant synapsin IIa by utilizing the baculovirus expression system. Rat synapsin IIa cDNA was introduced into the baculovirus genome via homologous recombination, and the recombinant baculovirus was purified. Spodoptera frugiperda (Sf9) cells infected with this virus expressed synapsin IIa as 5% of the total cellular protein. The recombinant protein was extracted from the particulate fraction of the infected Sf9 cells with salt and a nonionic detergent and purified by immunoaffinity chromatography. The purified synapsin IIa was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 0.8 mol of phosphate/mol of protein. Metabolic labeling with [32P]Pi demonstrated synapsin IIa phosphorylation in infected Sf9 cells. Using a homogenate of uninfected Sf9 cells, a cAMP-dependent protein kinase activity which can phosphorylate synapsin IIa was detected. Limited proteolysis of recombinant synapsin IIa phosphorylated in vitro and in vivo resulted in identical phosphopeptide maps. Further, synapsin IIa, like synapsin I, binds with high affinity in a saturable manner to synaptic vesicles purified from rat cortex.
Siow
Y L
YL
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021.
Chilcote
T J
TJ
Benfenati
F
F
Greengard
P
P
Thiel
G
G
D000818:Animals; D016367:Baculoviridae; D002458:Cell Fractionation; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005822:Genetic Vectors; D066298:In Vitro Techniques; D009036:Moths; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins; D015183:Restriction Mapping; D016704:Synapsins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000378:metabolism
D011994:Recombinant Proteins; D016704:Synapsins