Cloning, over-expression and purification of Escherichia coli murC encoding UDP-N-acetylmuramate--L-alanine ligase

Abstract

Gene murC encodes Uridine diphosphate N-acetylmuramate–L-alanine ligase, a 53 kDa enzyme involved in synthesizing the bacterial cell wall. The present study is designed to clone, overexpress and purify the recombinant murC using Escherichia coli as a model system. murC nucleotide sequence was obtained from the NCBI Gene database. The murC ORF from the genomic DNA of E. coli was amplified using primers containing restriction sites and a poly his-tag included in the pET 28b expression vector and was cloned into it. In E. coli BL21(DE3), the recombinant plasmid was transformed and overexpressed. The overexpressed recombinant His-tagged protein was purified using an affinity nickel column in a single chromatographic step. Since MurC ligases are a crucial enzyme involved in the synthesis of the bacterial cell wall and since it is absent in vertebrates including humans, this enzyme could be a desirable target for the development of antibacterial drugs. This method could be used for the production of MurC proteins that could help in identifying and screening small molecule inhibitors against pathogenic bacteria.