Expression of Putative RNase E from the Bacteriophage Shival

Shival is a novel bacteriophage that specifically infects Mycobacterium smegmatis to fully complete its replication cycle. Gene 68, which is part of the bacteriophage Shival's genome, has been characterized to code for the RNase E protein due to similarities between the base pairs in this gene and those usually conserved in RNase E genes. However, further scientific investigation is needed to make a conclusive statement about its identity. To that end, the gene was isolated and amplified via PCR and it was ligated into the pGEX vector. Gene sequencing was carried out to verify if any base pair aberrations had occurred in the gene during plasmid engineering. Then, BL21 E.coli strains were transformed with the plasmid and were induced to over-express the RNase E protein fused with Glutathione S-transferase (GST). To purify the protein, affinity chromatography was performed exploiting GST 's strong binding affinity for its reduced counterpart, GSH. Functions of other purified RNase E proteins range from mRNA degradation to tRNA maturation, which dramatically influence a cell's structure and life cycle. Therefore, determining the function of gene 68 can contribute to having a more thorough understanding of the bacteriophage's composition or infection.