Cloning And Expression Of Human Thrombospondin-1 CDNA Fragments

Thrombospondin-1 (TSP-1) is a large, trimeric, modular glycoprotein that is a major constituent of platelet a granules. TSP-1 is also secreted by a wide variety of epithelial and mesenchymal cells in patterns that reflect developmental changes in the embryo and response to injury in the adult. TSP-1 has been reported to have a lot of functions, such as inhibit growth and angiogenesis. The anti-angiogenesis property locates mainly on procollagen homology domains and type I repeats (properdin) in TSP-1. In this study two pairs of primers were designed and used to amplify TSP-1-1 (procollagen homology domains and type I repeats) and TSP-1-2(type I repeats) cDNA fragments which were identified by sequence analyses. Then these two cDNA fragments were expressed in Escherichia coli and Pichia Pastoris and their products were purified. The primary results were described as follows:1. Two cDNA fragments, named TSP-1-1 and TSP-1-2, were cloned by using two pairs of primers and PCR from human blood cells. Sequence analyses showed that sizes of those two fragments were 699bp and 528bp, respectively.2. The two fragments were inserted into pET-29a vector and transformed into E. coli JM109. Target peptides were found in inclusion body after IPTG induction. Soluble peptides were obtained by dissolving inclusion body in urea. The molecular weight of TSP-1-1 and TSP-1-2 is 30kD and20kD, respectively. TSP-1-2 peptides was further purified by CM-Sepharose chromatography.3. The two fragments were also cloned into pICZ a A vector and transformed into Pichia pastoris X-33. TSP-1-2 peptides was secreted into medium after methanol induction and its molecular weight is 20kD.