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Expression and initial characterization of recombinant mouse thrombospondin 1 and thrombospondin 3
Author(s) -
Chen Hui,
Aeschlimann Daniel,
Nowlen Julie,
Mosher Deane F.
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00460-7
Subject(s) - pentamer , spodoptera , thrombospondin , recombinant dna , protein subunit , microbiology and biotechnology , thrombospondins , biology , chemistry , thrombospondin 1 , biochemistry , genetics , gene , enzyme , metalloproteinase , angiogenesis
To analyze the function of TSP family members, we have expressed and purified mouse TSPI 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 μg, respectively, per ml of conditioned medium. Mature, secreted mouse TSPI and TSP3 had the previously predicted NH 2 ‐terminal sequences of DHVKDTSFDLFSI, and SQDLQVIDLLT, respectively. Analysis by SDS‐PAGE and rotary shadowing electron microscopy indicated that TSPI and TSP2 are disulfide bonded trimers whereas TSP3 is a disulfide‐bonded pentamer. Binding assays with 45 Ca 2+ 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.