Cloning of a Novel Centrin Target Peptide

Abstract

The origin of many disease states has been linked to genetic mutation, defects in gene expression, nuclear excision repair and ribosome biogenesis. Centrin, a calcium binding protein, has recently been found to regulate some of these processes along other target proteins within the nucleus. One target, Krr1, contains a K homology (KH) domain; which has been identified as a nucleic acid recognition motif, required for proper processing of pre-rRNA, for synthesis of 18S rRNA, and for the assembly of the 40S subunit. Our initial findings have identified a putative centrin binding site located within the KH domain of Krr1 within the Homo sapiens (Hs) centrin 2 (Hscen2-HsKrr1 complex) using bioinformatics tools. In this study, the KH domain (192 bp) was amplified by PCR and then ligated to the expression vector pET100 which adds a His tag to the peptide. Colony PCR was performed to identify the E. coli colonies that have been transformed effectively with the desired recombinant. The KH domain was then expressed in E. coli cells. To identify the presence of this peptide in the bacteria, an SDS-PAGE was performed. The overexpressed KH domain peptide will be purified and used for interaction studies with Hscen2.