Purification of myristoylated and nonmyristoylated neuronal calcium sensor-1 using single-step hydrophobic interaction chromatography.
Neuronal calcium sensors (NCSs) belong to a family of Ca(2+)-binding proteins, which serve important functions in neurotransmission, and are highly conserved from yeast to humans. Overexpression of the neuronal calcium sensor-1, called frequenin in the fruit fly and in frog, increases the release of neurotransmitters. Studying the functional role of frequenin in mammals and understanding its structural dynamics is critically dependent on the availability of active purified protein. Neuronal calcium sensors like other members of the family share common structural features: they contain four EF-hands as potential binding sites for Ca(2+) and an N-terminal consensus sequence for myristoylation. Previously, recoverin, distantly related to NCSs, has been expressed and purified from Escherichia coli, involving a combination of different chromatographic steps. NCS-1 has earlier been purified adopting a two-step procedure used for recoverin purification. We have overexpressed NCS-1 from rat in its myristoylated and nonmyristoylated form in E. coli and purified it from crude lysates using a single-step hydrophobic interaction chromatography. The purified protein was identified by Western blotting and mass spectrometry and assayed for its ability to bind Ca(2+) using a Ca(2+) shift assay, terbium fluorescence, and Stains-all binding. The present protocol provides a rapid, more efficient and simplified, single-step method for purifying NCS-1 for structural and functional studies. This method can also be applied to purify related proteins of the superfamily.
Fisher
J R
JR
Tosohaas, 156 Keystone, Montgomeryville, Pennsylvania, 18936, USA.
Sharma
Y
Y
Iuliano
S
S
Piccioti
R A
RA
Krylov
D
D
Hurley
J
J
Roder
J
J
Jeromin
A
A
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D019814:Myristic Acid / Q000378:metabolism*; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D002135:Calcium-Binding Proteins; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D011994:Recombinant Proteins; C101464:frequenin calcium sensor proteins; D019814:Myristic Acid; D002118:Calcium