Prokaryotic Expression Of Caspase-3 And Screening Of Its Inhibitors From Traditional Chinese Medicines

Apoptosis, also known as programmed cell death, has a strict regulatory mechanism. It has the characteristic of nuclear shrinkage, DNA fragmentation, cytochrome c release and apoptotic body formation, disappearing until engulfed by macrophages and so on. Apoptosis is mediated by three pathways. First, death receptor-mediated exogenous pathway, such as tumor necrosis factor, nerve growth factor mediated pathways. For some stimulation, endogenous Fas, TNFR associated with death domain FADD, TRADD produce direct or indirect effect to activate the pro-caspase. Second, mitochondrial-mediated endogenous pathway, when stimulated by apoptotic signals, mitochondria release cytochrome c into the cytoplasm to activate pro-caspase-9. The third pathway is the recently discovered endoplasmic reticulum pathway. Under stress situations, because of Ca2+ balance disorder in endoplasmic reticulum, activated calcium-dependent protein kinase can directly activate caspase-12. In addition, ER stress can also cause the relocation of cytoplasm caspase-7 and Bim to ER surface, activate caspase-12 and cascade reactions occur. Each of these pathways can cause a common final executed phase of apoptosis, that is through proteolysis, inactive proenzyme forms of caspase-3, -7 are activated, cascade reactions occur, and eventually induce apoptosis.Apoptosis can not only play a role in normal cells, but also in many diseases. Recent studies have shown that, many diseases, such as neurodegenerative diseases, cardiovascular diseases, diabetes and autoimmune diseases, are associated with apoptosis. These diseases are mainly caused by apoptosis of neurons and myocardial cells. Although the mechanisms of these diseases have not been deeply investigated, it is speculated that inhibition of apoptosis can become our new strategy of treating these diseases.Caspase family, especially caspase-3 as the key factor that executes apoptosis, play an important role in apoptosis-related diseases. It will undoubtedly become a new target for disease research. Caspase family belongs to cysteine aspartate-specific protease family, can specially cleavage proteins at aspartate sites to activate or cleavage proteins, thus execute the apoptosis process. In cells, caspases exist as inactive zymogen, and become active only when the large and small subunits form a heterodimer, then fold into a heterotetramer. Now many researchers have reported expression of recombinant caspases, and the strategies of prokaryotic expression of caspases can be divided into four: caspases are expressed as inclusion bodies, then dialyzed against acetic acid, refolded, ultrafiltrated, and finally we get active proteins; recombinant proteins are expressed as soluble forms, but they still require activation steps before they are active; rearrangement of large and small subunits, this strategy does not need processing or self-activation process; large and small subunits are expressed respectively, and then mixed with a certain concentration to become active. But for the expression of caspase-3, in addition to eukaryotic expression in Pichia, prokaryotic expression of caspase-3 is mainly through rearrangement of the large and small subunits, the protein can be active without any cleavage. Therefore, prokaryotic expression of recombinant caspase-3 becomes an important part of my paper. We want to get active caspase-3 recombinant protein for screening of inhibitors in vitro.Caspase inhibitors mainly include synthetic peptide and non-peptide inhibitors, but because of large molecular weight, strong hydrophobicity and poor permeability in clinical, the inhibitors always show strong toxic against cells at the the inhibitory concentration, causing the abnormalities of liver. So it is very promising to screening inhibitors from natural products. Traditional Chinese medicine (TCM) is of great significance in the Chinese medicine theory in our country, and is also an important resource for drug development. However, the ingredients of Chinese medicine are complex, the active ingredients of Chinese medicine are unclear, so the molecular mechanisms for the therapeutic effects of TCM have not been completely identified. Therefore, the study of the molecular mechanism of TCM is of great significance.The present paper's aim is through successfully expression of active recombinant casspase-3, characterization of the enzyme and screening of caspase-3 inhibitors from natural products in vitro, developing new drugs against caspase-3. First, we construct a recombinant plasmid which includes the gene of the truncated form of caspase-3, mainly large and small subunits. Then using different expression hosts to express the protein in soluble form under low-temperature conditions, and find E.coli BLP is the best host for the soluble expression. Then the histidine-tagged recombinant protein is purified by metal chelate chromatography. Through microplate reader, we determinate the activity of caspase-3, it is shown that we get the recombinant protein with high activity. Also we characterize its enzymatic properties, and determine the Km value and the IC50 for the specific inhibitor Ac-DEVD-CHO, these provide work basis for the inhibitor screening. Then, we screening the caspase-3 inhibitors from natural products using the fluorescent substrate method, finally we get two kinds of TCM (A & B) which can obviously inhibit the activity of caspase-3, with IC50 are respectively.In summary, we successfully construct recombinant caspase-3 with high activity, and it can be used for in vitro inhibitor screening. This provides a new way for the treatment of apoptosis-related disease, and also provides work basis for the development of new Drug.