Abstract：
: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.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Bjørndal B,Trave G,Hageberg I,Lillehaug JR,Raae AJdoi
10.1016/s1046-5928(03)00135-9keywords:
subject
Has Abstractpub_date
2003-09-01 00:00:00pages
47-55issue
1eissn
1046-5928issn
1096-0279pii
S1046592803001359journal_volume
31pub_type
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