Establish a PinPoint Xa protein purification system for analysis of Disabled-2-interacting proteins

• Main Authors: Yu-Lei Chang, 張玉蕾
• Other Authors: Ching-Ping Tseng
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/98494063939251167262

碩士 === 長庚大學 === 醫學生物技術研究所 === 95 === Disabled-2 (DAB2) is an adaptor protein that plays an important role in the regulation of megakaryocytic differentiation of K562 cells and nerve growth factor-induced neurite outgrowth of PC12 cells. We wanted to know the unknown interacting proteins which interact with DAB2 during cell proliferation and differentiation. Hence we established the DAB2 truncated form protein-ΔB (ΔB is a truncated form that lacked DPF motif and PRD domain of DAB2) purification system using the PinPoint Xa Protein Purification system. This system is used to produce and purify fusion proteins that are biotinylated in vivo. Biotinylated ΔB fusion protein was produced in soluble form in E.coli. The biotinylated ΔB fusion protein was purified by NeutrAvidin resins and was used to capture DAB2-interacting protein from K562 and PC12 cell lysates. Then DAB2-interacting proteins were separated from biotinylated ΔB fusion protein by high salt elution buffer. The elution condition did not affect biotin-avidin interaction of the columns and those columns could be regenerated. We analyzed the unknown interacting protein by SDS-PAGE and mass spectrometry. Taken together, we first established the PinPoint Xa Protein Purification system. We found lactamase、yippee-like-3、myosin VI、mitofusin-1、sepiapterin reductase and glutamyl aminopeptidase might be DAB2-interacting proteins.