Molecular cloning and homology modelling of human Cyclin Dependent Kinase 3 (CDK3)

Abstract

Cancer comprises of a set of over 100 diseases that each developed in time while involving the unrestrained division of body cells. It disrupts the cell cycle in which it forces the cells to propagate irrepressibly. Cyclin Dependent Kinases (CDKs), a group of over more than 20 members are referred to as “master regulators” of progression of cell cycle, molecular engines that activate cell cycle transitions. CDK3, a member of this family, is an important component of cell cycle regulation and is also a part of the cell cycle transitions of G0 G1 and G1 S stages. Over-expression of CDK3 in many cancer cell lines, indicated that it may have an important role in malignant transformation and cell proliferation. The human CDK3 gene codes for a 307-amino acid protein. In this study, the gene coding for CDK3 was amplified from MCF-7 bBreast cancer cell line cDNA and cloned into cloning plasmid pGEM T-easy. The amplified gene has a size of 915 base pairs. The sequence was verified by Sanger sequencing and pairwise sequence alignment with Uniprot reference sequence (Q00526). It was then cloned into pGEM®-T EASY cloning vector by direct ligation of PCR product with linearized 3’-A overhangs. The three-dimensional (3D) structure of Cyclin Dependent Kinase 3 (CDK3) was modelled based on the crystal structure of Cyclin Dependent Kinase 2 (CDK2). Prediction of 3-D structures of CDK3 is a stepping stone towards further analysis of its function and potential. It shed light on various possible actions of this enzyme based on the predicted folding of the model, especially in terms of binding affinities, paving the path for further favorable modifications. This study clearly predicted that the conserved catalytic residues and domains characteristic which provides a preliminary insight into the specific function of this enzyme. Further experimental studies are required to characterize and analyze the actual functional capabilities of this enzyme.