Attempts to Develop a Recombinant Protein with Antioxidant and Thrombol Ytic Activity - In-Vitro Study
Author(s): C.Revathy , A.J. Manjula Devi *
The objectives were framed bearing in mind to generate a synergetic protein of higher efficacy so as to handle both the thrombotic status and ischemia mediated free radical injury to fight Myocardial infarction. Amplification of human Thioredoxin gene from lung cancer cell line A549.Cloning of human Streptokinase in pRSET A at Barn HI and Eco RI site and fusion of human thioredoxin in the recombinant clone pRSET A/Sk at Ndel and Barn HI site In this study, the human thioredoxin isolated from lung cancer cell lines is fused with streptokinase to generate a fusion construct so as to develop a synergetic protein of higher efficacy in the thrombolytic and the antioxidant activity. Expression studies and solubility of the recombinant protein was studied., Maximum soluble fraction achieved by optimizing various parameters like host strain, optical density, post induction temperature, inducer concentration etc... was subjected to ion exchange chromatography obtaining a sing1e band corresponding to the molecular weight in SDS-PAGE and confirmed by western blot as well. Activity of the purified recombinant TRX-Sk protien was confirmed using in-vitro biological assay such as: Insulin reduction assay - Retention of the biological activity of Thioredoxin in the recombinant protein in comparison with the control.