In vitro phosphorylation of mouse osteopontin expressed in E. coli.
To understand the role of post-translational modifications on the structure and function of osteopontin, a secreted glycosylated phosphoprotein, we expressed mouse osteopontin in E. coli as a fusion protein with glutathione-S-transferase (GST). The purified fusion protein was cleaved by factor Xa generating GST (26 kDa) and recombinant osteopontin (60 kDa). The fusion protein was phosphorylated in vitro by cytosolic, microsomal, and casein kinase II fractions from mouse kidney homogenates. The fusion protein and recombinant osteopontin were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. The suitability of the fusion and recombinant proteins as model substrates for the study of the function(s) and post-translational modifications of osteopontin is discussed.
Ashkar
S
S
Laboratory for the Study of Skeletal Disorders and Rehabilitation, Harvard Medical School, Children's Hospital, Boston MA 02115.
Teplow
D B
DB
Glimcher
M J
MJ
Saavedra
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D053495:Osteopontin; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012795:Sialoglycoproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; D053495:Osteopontin; D005982:Glutathione Transferase