Expression and purification of receptor for activated C-kinase 1 (RACK1).
Receptor for activated C-kinase (RACK1) binds to protein kinase C and functions as an anchor for several other cellular components. Most in vitro studies of RACK1 have been carried out with RACK1 fused to a soluble fusion protein partner, such as GST or MBP. Here, we show that fusion complexes may exist as large soluble aggregates and thereby lead to false conclusions about the biological activity of RACK1. We developed a purification procedure that gave soluble monodisperse molecules of the protein. The RACK1 gene was cloned and expressed in a pMAL vector. After purification of the resulting MBP-RACK1 fusion protein, RACK1 was excised from MBP by thrombin, rendering RACK1 in a soluble monodisperse form as monitored by fluorimetric static light scattering, gel filtration, and ultracentrifugation. Circular dichroism analysis revealed that RACK1 was properly folded with a T(m) of approximately 62 degrees C and contained the predicted portions of secondary structures. The biological activity of the purified protein was verified by binding to activated protein kinase C. The production of soluble, high-purity RACK1 will allow structural studies and functional in vitro studies to identify interacting partners to this important scaffold protein.
Bjørndal
Bodil
B
Department of Molecular Biology, University of Bergen, HIB, P.O. Box 7800, 5020, Bergen, Norway.
Trave
Gilles
G
Hageberg
Ingeborg
I
Lillehaug
Johan R
JR
Raae
Arnt J
AJ
D000688:Amylose / Q000737:chemistry; D026481:Blotting, Far-Western; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D019169:Jurkat Cells; D058619:Maltose-Binding Proteins; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000378:metabolism; D014461:Ultracentrifugation
D002352:Carrier Proteins; D020536:Enzyme Activators; D058619:Maltose-Binding Proteins; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D000688:Amylose; D011493:Protein Kinase C; D013917:Thrombin