Expression and initial characterization of recombinant mouse thrombospondin 1 and thrombospondin 3.
To analyze the function of TSP family members, we have expressed and purified mouse TSP1 and TSP3 encoded by recombinant baculoviruses in Spodoptera frugiperda cells and compared these TSPs to mouse TSP2 prepared in a similar way. Yields of purified TSP1 and TSP3 were 5-15 and 2-4 micrograms, respectively, per ml of conditioned medium. Mature, secreted mouse T41 and TSP3 had the previously predicted NH2-terminal sequences of DHVKDTSFDLFSI, and SQDLQVIDLLT, respectively. Analysis by SDS-PAGE and rotary shadowing electron microscopy indicated that TSP1 and TSP2 are disulfide bonded trimers whereas TSP3 is a disulfide-bonded pentamer. Binding assays with 45Ca2+ as ligand and immobilized TSP1, TSP2 and TSP3 demonstrated that all three TSPs are calcium-binding proteins. These results are consistent with previous studies of TSP structure and demonstrate that Spodoptera cells process and secrete TSPs having the same subunit organizations and structure as the native vertebrate molecules.
Chen
H
H
Department of Biomolecular Chemistry, University of Wisconsin, Madison, 53076, USA.
Aeschlimann
D
D
Nowlen
J
J
Mosher
D F
DF
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera / Q000166:cytology; D019699:Thrombospondins
D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D019699:Thrombospondins