CLONING, EXPRESSION AND in vitro ACTIVITY OF FUSION PROTEINS: ENDOSTATIN-PRO-APOPTOTIC PEPTIDES
Chura-Chambi, R.M., Lino, F.S.O., Pietro-da-Silva, A., Malavasi, N. V., Rodrigues, D.B., and Morganti, L.
Instituto de Pesquisas Energéticas e Nucleares – IPEN – CNEN/SP
Endostatin, a 20 kDa COOH-terminal fragment of collagen XVIII, has been shown to act as an anti-angiogenic agent which specifically inhibits proliferation of endothelial cells and growth of various primary tumors. In the present study we report the expression and in vitro activity of proteins composed of two functional domains: endostatin, that presents affinity for activated endothelial cells, used to guide the fusion protein and allow its internalization by the targeted cells, and a second domain composed by short peptides (16aminoacids), corresponding to the minimal sequence of BH3 domains of pro-apoptotic proteins required to promote apoptosis. These proteins should be more efficient in the treatment of mice tumors than the isolated endostatin.
Objective
Clone and express fusion proteins which present the endostatin advantages of internalization by activated endothelial tumor cells and higher degree of apoptosis induction after internalization.
Methods
The pro-apoptotic peptides used in this study were Bax and Bak. The insertion of sequences coding the minimal sequence of the BH3 domain of pro-apoptotic peptides Bax and Bak into the expression vector pET28-containing endostatin gene was performed using site-specific mutagenesis. The fusion proteins, endo-Bax and endo-Bak, were obtained as insoluble cytoplasmic inclusion bodies, expressed in E. coli. BL21(DE3). The expression was analyzed by SDS-PAGE/Western blot assay. The proteins were solubilized and purified by immobilized metal chelate chromatography. The biological activity of the fusion proteins was measured through detection of the viability of Human Umbilical Vein Endothelial Cells (HUVE-EC) using the MTS dye.
Results
Mutations of the vectors presenting the apoptotic domains endo-Bax and endo-Bak were obtained and confirmed by DNA sequencing. SDS-PAGE and Western blot analysis confirmed the expression of endostatin and the fusion proteins, endo-Bax and endo-Bak. The assay on HUVE-EC-C cells demonstrated that the fusion protein endo-bax presented a higher effect on the decrease of viability of the cells (45.0%) than endo-bak (17.4%) or wt endostatin (16.1%).
Conclusions
The fusion proteins, endo-Bax and endo-Bak were successfully obtained; their expression was confirmed by Western blot analysis. The viability assay with HUVE-EC-C cells showed that end-bax and end-bak were more potent than wt endostatin.
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