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Optimization of Refolding and Purification of a Novel Recombinant Human Endostatin from E.coli

Author: ZhaoLiXi
Tutor: JiangYongPing；DaiWei；DingXinXin
School: Peking Union Medical College , China
Course: Biomedical Engineering
Keywords: Recombinant human endostatin Renaturation Purification Chick chorioallantoic membrane assay
CLC: R346
Type: Master's thesis
Year: 2010
Downloads: 37
Quote: 0
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Abstract


Objective: To optimize the refolding of recombinant human vascular endothelial growth inhibin (rhED) program , in order to improve its refolding yield , and after initial detection renaturation rhED biological activity of anti-angiogenic further study and utilization of rhED lot of there are activity rhED protein . Methods: a pQE3 with rhED cDNA expression vector to transfect E. coli E.ColiM15 IPTG induction fermentation produce rhED inclusion bodies and extract rhED inclusion bodies . Refolding , first with 6mol / L guanidine hydrochloride dissolved after extraction rhED inclusion body protein solution is diluted , in a suitable concentration of urea and stirred for a long enough period of time , combined stepwise dialysis method for the renaturation , to identify the main impact of the refolding factor , and optimize optimal refolding conditions . Then, using the standard curve method to measure the the concentration of rhED , authentication and calculating of the SDS -PAGE and renaturation yield . Cation exchange chromatography after the end of the refolding purified rhED and concentrated by ultrafiltration to the appropriate concentration . Finally, with rhED specific monoclonal antibody and chick chorioallantoic membrane inhibit blood vessel growth assay refolded protein activity . Results : 1 L of rhED bacteria fermentation broth can produce 15g rhED of inclusion bodies . By optimizing the refolding conditions rhED renaturation rate can reach 46%. The three most important influencing factors of the complex - renatured protein solution concentration , urea concentration and pH value . Refolding protein concentration of 0.3mg/ml, and the urea concentration of 3.5mol / L and a pH of 8 is suitable refolding conditions . Good cation exchange chromatography purified refolded rhED. Renaturation purified rhED can with rhED -specific monoclonal antibody reaction , and inhibit the growth of blood vessels experimental activity shown to inhibit the growth of blood vessels in the chick chorioallantoic membrane . Conclusion : At this point refolding conditions improve rhED renaturation rate has been established, the renaturation program will greatly promote rhED the pre-clinical studies and clinical research .

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CLC: > Medicine, health > Basic Medical > Human biochemistry, molecular biology
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