Abstract
The p21 (Waf1/Cip1) protein is a general inhibitor of cyclin dependent kinase (CDK). Although it has been known that p21 interacts with CDK, cyclin D1 and proliferating cell nuclear antigen (PCNA), recent studies have found that p21 interacts with many other proteins. For example, p21 can interact with procaspase-3 to inhibit caspase-3 activation. The aims of the study are two; to obtain enough p21 protein for structural analysis, and, to explore the novel p21 interacting proteins. A large quantity of recombinant human p21 protein was obtained from E. coli. Although the proteins were initially collected in the form of inclusion bodies, they were renatured through a published scheme. Circular dichroism spectroscopy shows that the refolded proteins acquired helical structure at the similar fraction as predicted from the primary sequence of the protein. Immunoprecipitation and Far-Western analyses show that the refolded p21 protein bound with PCNA, CDK2, and cyclin D1. Some proteins of the CHO-K1 cells were found to interact with the p21 protein, although their identities remain unknown. These results suggest that the recombinant p21 protein is useful to pursuit the aims described earlier.