BIỂU HIỆN VÀ TINH SẠCH NHÂN TỐ PHIÊN MÃ NF- κB P65 CỦA NGƯỜI SỬ DỤNG TẾ BÀO VẬT CHỦ E. coli ĐỊNH HƯỚNG ỨNG DỤNG SÀNG LỌC CHẤT ỨC CHẾ UNG THƯ

Abstract

Background: NF-κB p65 transcription factor is present in almost all human cell types and plays important role in regulating human immune system. Up-regulation of NF-κB p65 is associated with a number of human cancer types. In order to screen inhibitors of NF- κB p65 using in vitro approach, it is required to produce large amount of recombinant NF- κB p65 with high purity.  Objectives: 1. To design expression construct that can express human NF- κB p65 in E.coli host cells, 2. To purify recombinant NF- κB p65 with high purity. Materials and methods:  Codon optimization and DNA cloning methods were used to design expression vector containing gene encoding NF-κB p65 transcription factor. The recombinant protein was expressed in E. coli BL21(DE3) and then purified using nickel affinity chromatography. Results: The DNA fragment encoding human DNA binding domain (Rel homology domain) of NF-κB p65 transcription factor was successfully codon-optimized and inserted in to pET-28a expression vector to generate pET-28a/ NF-kB p65 construct. The pET-28a/ NF-κB p65 was then transformed in to BL21(DE3) E. coli competent cells using heat-shock method. The expression of recombinant NF-κB p65 protein in E. coli BL21(DE3) strain was successfully induced at both 20^oC and 37^oC with 100 µM IPTG. The expression level of recombinant NF-κB p65 was very high as 50 mg the recombinant protein was obtained per liter of LB culture. Recombinant NF-κB p65 was purified using nickel bead column with high purity. Conclusion: We have successfully expressed and purified recombinant NF-κB p65 with high yield of 50 mg/L. The obtained NF-κB p65 with high purity can be used for screening of potential drug for NF-κB p65-related cancer diseases.