Quality Control Of Recombinant Protein Tmsm And Antitumor Effects Study

The human Survivin gene was obtained by Altieri DC from RT-PCR method in1997in human breast cancer cells. The Survivin protein inhibitor of apoptosis proteins (Inhibitor of apoptosis protein, a member of the IAP) family, what containings70amino acids of the rod-shaped baculovirus IAP repeat sequence (Rod baculovirus the IAP repetitive sequence BIR). Survivin protein is rarely expressed in normal cells, and abundantly expressed in cancer cells, the unique nature is allowed to become a hotspot of cancer research. It can be used as a marker of poor prognosis in cancer treatment process, and it may also become a new anticancer target. Survivin protein mutant Survivin (T34A),is a dominant negative mutant of Survivin protein. It can reverse the role of Survivin of the anti-apoptotic, promote the death of cancer cells. Through to the limited capacity of the cell membrane, and therefore our laboratory in this mutant with Survivin penetrating peptide (Trans-activating transcriptional activator, TAT) TAT-Surivin (T34A) mutant gene is constructed on the basis of, in Escherichia coli expression of this protein, referred TmSm protein.The main content of the paper is TmSm protein expression, purification and quality and pharmacodynamic testing.(1)10L fermentor, bacterial wet weight up to160g, up to35.5%of the total bacterial protein expression levels of the target protein. E. coli fermentation broth with a high pressure homogenizer under pressure of800MPa crushing, and10,000rpm centrifuged to give40g of inclusion bodies, up to80%purity.(2) The use of30L of the fermentor, cell growth density of up to30.6OD. Fermentation the wet cells yield of27～31g/L, the purpose of the protein expression levels of30to32%of the total bacterial proteins. Repeated fermentation batches on a30L tank, control the process and the result is basically stable. Washing, purification and refolding of inclusion bodies, and ultimately obtained a purity of greater than95%of10g available for further experiments with TmSm protein.(3) On the basis of optimization of the purification process in accordance with the the requirements of the national pharmacopoeia TmSm the molecular weight of the protein,peptide mapping, C/N terminal amino acids, the UV absorption spectrum, RP-HPLC purity, host cell protein residues,endotoxin content, the outer the source DNA residual amount, rats hemolytic detection test, TmSm powder for injection immunogenicity testing, comply with the requirements of the State Pharmacopoeia.(4) Early cells in the laboratory experimental basis on five common tumor cell B-cap-37, MCF-7s, MCF7/ADR SW1990, A549proapoptotic experiments prove that the tumor cells have better proapoptotic effect.(5) On the basis of human breast cancer cell B-Cap-37cells in BALB/c-nu mice model had been established,we decided to do the mice preliminary pharmacodynamics test. After the test,we found that the size and weight of the tumor volume of TmSm experimental drug group was significantly less than the control group. Proved TmSm protein is capable of tumor cells of the effective destruction of the mouse model animals.