Regular Article
Expression, Purification, and Characterization of a Highly Soluble N-terminal-Truncated Form of the Neuron-Specific Membrane-Associated Phosphoprotein SCG10

https://doi.org/10.1006/prep.1996.0679Get rights and content

Abstract

SCG10 is a neuron-specific growth-associated protein with high sequence homology to the ubiquitous phosphoprotein stathmin/Op18. The main structural difference between the two proteins is the 34-amino-acid N-terminal extension of SCG10, which is responsible for the membrane attachment. Full length SCG10 has been purified and shows limited solubility, in contrast to stathmin, which is a highly soluble protein. In order to obtain a more soluble form of SCG10 which would be better suited for biochemical and structural studies, we deleted the N-terminal extension and expressed the C-terminal portion of the protein. Two forms of N-terminal-truncated SCG10 (ΔSCG10 and ΔSCG10r) were purified to homogeneity in a four-step purification procedure. ΔSCG10 starts at amino acid 35 and ΔSCG10r at amino acid 48 in the SCG10 sequence, giving proteins of 16,899 and 15,189 kDa, respectively. The truncated SCG10 was highly soluble up to concentrations of 20 mg/ml. The proteins were like the full length SCG10 substrate for serine/threonine protein kinases, including MAP kinase, PKA, and p34cdc2kinase. With these highly soluble forms of SCG10 biochemical and structural studies of this multiphosphoprotein become feasible.

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