Rapid purification, site-directed mutagenesis, and initial characterization of recombinant RC3/neurogranin.
RC3/Neurogranin is a postnatal-onset, forebrain-specific, thyroid hormone-regulated, protein kinase C (PKC) substrate that binds calmodulin (CaM) and accumulates in dendritic spines. We bacterially expressed and purified RC3 and, for comparison, GAP-43/neuromodulin to near homogeneity using relatively simple procedures. We then raised antisera against recombinant RC3 that does not crossreact with GAP-43 and is suitable for immunohistochemical analysis of brain slices. We also constructed over 30 RC3 sequence variants by PCR-mediated, site-directed mutagenesis, and purified four of these to near homogeneity. The elution profiles displayed by RC3 and sequence variants during purification on CaM-Sepharose columns suggest that two different affinity forms of the RC3.CaM complex coexist when Ca2+ is absent and that GAP-43.CaM interactions are far more sensitive to salt than those that occur between recombinant RC3 and CaM. Variant proteins in which serine 36 was changed failed to serve as a substrate for PKC, implicating this as the target residue.
Gerendasy
D D
DD
Department of Molecular Biology, Scripps Research Institute, La Jolla, CA 92037, USA.
Herron
S R
SR
Wong
K K
KK
Watson
J B
JB
Sutcliffe
J G
JG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004926:Escherichia coli; D019922:GAP-43 Protein; D006624:Hippocampus / Q000737:chemistry; D007106:Immune Sera; D008562:Membrane Glycoproteins / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D051579:Neurogranin; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016415:Sequence Alignment
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D019922:GAP-43 Protein; D007106:Immune Sera; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; C496651:Nrgn protein, rat; D011993:Recombinant Fusion Proteins; D051579:Neurogranin; D002118:Calcium