Purification and characterization of recombinant human p21 protein expressed in Escherichia coli

• Main Authors: Kuo-Pao Lai, 賴國寶
• Other Authors: Yin-Chang Liu
• Format: Others
• Language: en_US
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/57366859184311521845

碩士 === 國立清華大學 === 生命科學系 === 88 === Human p21 (Waf1/Cip1) protein is a potent inhibitor of cyclin dependent kinase (CDK).p21 (Waf1/Cip1) could bind to cyclin-cdk complex and inhibit the catalytic activity of cdk. The inhibitory effect of cdk could cause the cell cycle arrest and undergo repair events. p21 (Waf1/Cip1) could also bind to PCNA (Proliferating Cell Nuclear Antigen) via the C-terminal domains and inhibit PCNA-dependent replication and repair. The full-length cDNA of human p21 Waf1/Cip1 was cloned into a prokaryotic expression vector and transformed into E. coli BL21 (DE3) cells. Upon induction with isopropylthiogalactopyranoside (IPTG), the recombinant protein accumulated as insoluble inclusion bodies. The inclusion bodies were solubilized with 6 M urea or chemical lysis method (4.5 M urea and 0.1 Mβ-mercaptoethanol). The solubilized recombinant protein was purified by metal-affinity chromatography on Ni2+—column and used a imidazole gradient to elute the target protein. By comparing the different methods, we could obtain the purer recombinant protein by chemical lysis and affinity chromatography method. To characterize the activities of the purified recombinant human p21 (Waf1/Cip1) protein, we used ELISA assay to demonstrate that recombinant human p21 (Waf1/Cip1) protein could interact with PCNA in vitro. We also used immunoprecipitation (IPT) and immunoblotting assay to demonstrate the same properties. According to the previous studies, the p21 (Waf1/Cip1) protein could not be detected in CHO.K1 cells (Chinese Hamster Ovary) after UV irradiation. We found that some p21 (Waf1/Cip1) interaction components in CHO.K1 cells after UV irradiation by IPT assay. By this way, we could further investigate the components involved in the cell cycle regulation of CHO.K1 cells following UV irradiation.