Expression, purification, and characterization of a highly soluble N-terminal-truncated form of the neuron-specific membrane-associated phosphoprotein SCG10.
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 (delta SCG10 and delta SCG10r) were purified to homogeneity in a four-step purification procedure. delta SCG10 starts at amino acid 35 and delta SCG10r 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 p34cdc2 kinase. With these highly soluble forms of SCG10 biochemical and structural studies of this multiphosphoprotein become feasible.
Antonsson
B
B
Geneva Biomedical Research Institute, Switzerland. BEA6063@GGR.CO.UK
Montessuit
S
S
Di Paolo
G
G
Lutjens
R
R
Grenningloh
G
G
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins; D002847:Chromatography, Agarose; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D005822:Genetic Vectors / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D008868:Microtubule Proteins; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012995:Solubility; D017783:Superior Cervical Ganglion / Q000737:chemistry
D002352:Carrier Proteins; D008565:Membrane Proteins; D008868:Microtubule Proteins; D009414:Nerve Growth Factors; D010750:Phosphoproteins; D011994:Recombinant Proteins; C512848:Stmn2 protein, rat