Cloning of Methionine-adenosyl-transferaseGene in E.coli

摘要

Methionine-adenosyl-transferase is an enzyme which catalyses the synthesis of S-Adenosyl Methionine (SAMe) using methionine and ATP. It is also known as AdoMet which is well known methyl donor for the majority of methyl-transferases which modifies DNA, RNA, histones and other proteins, dictating replicational, transcriptional and translational fidelity, mismatch repair, chromatin modeling, epigenetic modifications and imprinting. The objective of the present work is to clone the Methionine-adenosyl-transferase gene in E.coli for further expression and characterization studies. The genomic DNA was isolated from E.coli. The OD26o/ OD280 value of the isolated DNA was found to be 1.8 which confirmed the purity of isolated genomic DNA. Suitable primers were designed in order to amplify the target gene through / PCR. Amplified genes were analyzed through Agarose Gel Electrophoresis. pBSK(+) a phagemid was used as a cloning vector in this work. The amplified gene product was purified and inserted in the multiple cloning site near the lacZ sequence in pBSK (+) cloning vector using EcoRV for restriction and T4 DNA ligase for ligation. The ligated vector was used for the transformation directly into E.coli DH5a. The transformed cells were allowed to grow inthe LB plate overnight with appropriate antibiotics in order to pick the blue and white colonies the next day. Colony PCR was performed to confirm the presence of gene of interest from the grown white colonies. Those recombinant cells containing the gene of interest in their plasmid were allowed grow in the LB broth with respective antibiotics in order to multiply the plasmid containing the gene of interest. The plasmid DNA was isolated and restricted by Hind III and Pst I and the insert was prepared using Hind III andNde I in order to prepare it for cloning and further expression studies in the pET 24a (+) expression vector.