Expression And Activation Research Of Human Kallistatin-sTRAIL Fusion Protein

Tumor, nowadays, is one of the most dangerous human diseases, the mortality ofcancer patients is very high. The preferred method of traditional treatment is surgery,for patients who are not suitable for surgery, radiotherapy, chemotherapy or acombination of both is the second choice. However, traditional treatments often killthe innocent which cause great side effect to patients.With the development of related disciplines in biology in recent years, some newconcepts and methods of cancer treatment have been proposed, such as gene therapy,immunotherapy, targeted therapy, apoptosis induction therapy and anti-angiogenictreatment. These biological cancer treatments to a certain extent can better alleviatethe suffering of cancer patients in clinical practice.According to the spirit of multi-target and multiple signaling pathways of tumorbiological therapy. In this paper, we selected two proteins which are all very hot incancer therapy: recombinant human Kallistatin and sTRAIL. Kallistatin is anendogenous protein with401amino acid composition, and molecular weight of58KD;sTRAIL is a soluble peptide and the extracellular domain of TRAIL (114-281aminoacid residues), and it also has complete biological activity of TRAIL and canspecifically combine with the natural receptors of TRAIL activating the signaltransduction pathway leading to apoptosis. Numerous studies show that Kallistatin isable to inhibit angiogenesis and vascular endothelial cells (HUVEC) proliferation andmigration, with a broad-spectrum anti-tumor activity. sTRAIL has targeted inducedapoptosis in tumor cells. In addition, the recombinant protein drug has been widelyused in clinical technology and the technologies are relatively mature, moreover,there is no reports about the fusion protein of Kallistatin and sTRAIL both at homeand abroad, so we built and expressd the fusion protein KT by means of geneticengineering, and through the initial anti-tumor experimental study, we found that thisfusion protein does have a certain degree activity of Kallistatin and sTRAIL.At the begining, through the literature review we found that there were manyinadequates to prokaryotic engineered bacteria(E.coli et cl.) to express this twoproteins, such as being easy to produce inclusion bodies, poor protein activity or low expression efficiency, however, eukaryotic expression systems may be well to solvethese problems, so we decided to use Pichia pastoris GS115to expressed this fusionprotein.At first, we designed appropriate primers to build the pPIC9-Kallistatin-sTRAILeukaryotic expression vector, and then imported it to pastoris GS115by electricalconversion, what`s more, after pilot-scale fermentation in5L fermentor, we purifiedthe fermentation broth through PHENYL SEPHAROSETM6FAST FLOW, SephadexG-25and Heparin Sepharose-6gel column. SDS-PAGE electrophoresis showed thatthere was little of hybrid protein in the purified protein and the protein purityachieved98%. The protein concentration reached0.672mg/ml detected by ELISA.It Is worthy to point out that we introduced the RGD peptide (containing arginine-glycine-aspartic acid peptide) to connect the two proteins, and the protein sequenceof RGD we used is GGGGSG-RGD-SGGGG. This short peptide can be recognizedby integrin and some ligand recognition sites. Tumor cells and the newly generatedblood vessels can selectively express certain integrins (such as αvβ3or αvβ5), andthus can combined with RGD peptide, which qualified KT protein the characteristicof tumor and neovascular targeting. For anti-angiogenic, anti-cell proliferation andmigration activity of Kallistatin, we designed in vitro anti-angiogenesis experiment,in vitro inhibition of HUVEC, Hela and NCI-H446cell proliferation and inhibition ofHUVEC migration experiments. The results showed that, the20μg/ml KT protein wasable to inhibite HUVEC to generate a small tube, and the inhibition rate reached morethan60%. Effective inhibition concentration of KT protein on HUVEC, Hela andNCI-H446cell proliferation is16μg/ml,32μg/ml and16μg/ml, inhibition rates were32%,38%and17%respectively.13.5μg/ml KT protein could inhibit HUVECmigration. For the promoting tumor cell apoptosis ctivity of sTRAL, we use bothHela and NCI-H446tumor cells to detect this activity of KT. After induceing tumorcells by KT, The Hoechst staining results showed that apoptotic phenomenon werevery remarkable.The preliminary experiments showed that the KT fusion protein to a certain extenthas the double activity of Kallistatin and sTRAIL which provided certain reference toexplore future applications of recombinant proteins and tumor multi-target, targeted therapies.