Isolation, Purification, Modeling, and binding site analysis of the Salmonella Bacteriophage ɛ34 repressor for subsequent crystallization and structure‐function studies

Abstract

The bacteriophage ɛ34 is one of many phages that infect the bacteria Salmonella which is the main cause of salmonellosis (food poisoning). The bacteriophage ɛ34 encodes a protein called the cI repressor that is like other phage repressors. It has a C‐terminal protease domain which is about 80% identical to the repressors of phage λ (which infects E. coli), but its DNA binding N‐terminal portion is only about 50% identical to its closest known relatives in phages λ, and Lex A. This means that the ɛ34 repressor may have the same pathway for inactivation as phage λ, but probably has a different operator binding specificity due to lower sequence homology of its DNA‐binding domain. The N‐terminal DNA‐binding domain is homologous to the Helix‐Turn‐Helix family of DNA‐binding domains.The main goal of this project was to initiate a structure‐function study on the repressor protein of phage ɛ34. The ɛ34 Bacteriophage DNA was first isolated and purified. The gene encoding the bacteriophage ɛ34 repressor was amplified by PCR and cloned into a pET11a vector. The cI repressor, was overexpressed and purified by immobilized metal affinity chromatography and ion exchange chromatography. We also generated a homology model of this protein using the bacteriophage λ as a template in the SWISS‐MODEL homology modelling server. Our models predict a unique structure for the N‐terminal Helix‐Turn‐Helix domain. Finally, we have identified a potential binding site upstream of the cI gene on the Bacteriophage λ DNA. Crystallization trials are currently in progress. This project was done as a collaborative venture between two laboratories, Huntingdon College (Dr. Doba Jackson) and Alabama State University (laboratory of Dr. Robert Villafane). This project is in line with Dr. Robert Villafane's research into the Genomic analysis of bacteriophage ɛ34 of Salmonella enteric serovar Anatum.Support or Funding Information1Department of Chemistry & Biochemistry, Huntingdon College2Department of Biology, Alabama State UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.