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Preparation and Functional Analysis of Recombinant Human Smac and Smac-PTD

Author: HuYing
Tutor: HeFengTian
School: Third Military Medical University
Course: Biochemistry and Molecular Biology
Keywords: Second mitochondrial activator of caspase The protein transduction domain cDNA cloning Apoptosis Prokaryotic expression Protein purification Tumor
CLC: Q78
Type: Master's thesis
Year: 2006
Downloads: 51
Quote: 0
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Abstract


Smac/DIABLO (second mitochondria-derived activator of caspase or direct IAP binding protein with low pI) is a protein released from mitochondria in response to apoptotic stimuli along with cytochrome c, which promotes caspase activation in the cytochrome c/Apaf-1/caspase-9 pathway and functions as a direct endogenous inhibitor of IAP proteins. Smac is translated as a 239 residues precursor protein with a mitochondrial targeting sequence at its N-terminus that is removed upon import. Although it resides within the intermembrane space of mitochondria in healthy cells, upon cellular stress such as UV irradiation, DNA damage and growth factor depletion Smac is released into the cytosol where it acts as a sensitizer for caspase acting by binding to inhibitor of apoptosis proteins (IAP) via a short N-terminal sequence that disrupts binding of caspase to the IAP BIR3 domain. Smac act as a key apoptosis regulator and have become an attractive molecular for treating a variety of human tumors.Like cytochrome c, Smac/DIABLO delineate their functions in cytosol, but the macromolecules that we have got in lab could not import into cytosol and this feature limits their applications in cancer therapy. Whereas, the protein transduction domain (PTD) from the HIV-1 TAT protein can retrieve this flaw. PTD is an 11 amino acid arginine rich peptide with the ability to rapidly translocate into cells both in vivo and in vitro. Fusion of PTD with proteins and peptides has been shown to facilitate an effective transduction of the fused cargos into cultured cells and animal tissues while preserving their biological activity. PTD-Fusion proteins prepared have been shown to transduce into a variety of both primary and transformed cell types. Moreover, PTD-fusion proteins were shown to transduce into all cells and tissues present in mice. To date, this strategy has resulted in the production of more than 60 full-length proteins with functional domains from 15 to 120 kDa. So we prepared the hSmac and hSmac-PTD that may pave the way for further studies.So firstly in our experiments, the gene sequence encoding the 56-239 amino acids of Smac (mature Smac) was cloned by RT-PCR and Smac-PTD was constructed by three times

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