85 Cloning, Expression, Purification and Crystallization of a Lysozyme Encoded by Bacteriophage SN

Wednesday, November 4, 2009: 9:40 AM
Kohlberg (Camino Real Hotel)
Nadia Herrera , Department of Chemistry, The University of Texas at El Paso, El Paso, TX
Maxim Filchikov , Laboratory of Molecular Bioengineering, Russian Academy of the Sciences, Moscow, Russia
Zacariah L. Hildenbrand , Department of Chemistry, The University of Texas at El Paso, El Paso, TX
Sudheer K. Molugu , Department of Chemistry, The University of Texas at El Paso, El Paso, TX
Vadim V. Mesyanzhinov , Laboratory of Molecular Bioengineering, Russian Academy of the Sciences, Moscow, Russia
Konstantin Miroshnikov , Laboratory of Molecular Bioengineering, Russian Academy of the Sciences, Moscow, Russia
Ricardo A. Bernal , Department of Chemistry, The University of Texas at El Paso, El Paso, TX
            The bacteriophage SN is a virulent phage that infects the bacterium Pseudomonas aeruginosa.  It was isolated from Lake Chernoe in the Vladimir region of Russia and is related to the PB1-like species of the Myoviridae family.  The DNA genome is composed of 66,391 base pairs, has 89 predicted open reading frames, and encodes more than 20 structural proteins. One of the open reading frames of this newly discovered bacteriophage has high sequence identity to other lysozyme and chitinase genes in the GenBank database.  It is therefore assumed that this putative lysozyme encoded by bacteriophage SN is utilized for digestion of the host cell wall prior to injection of the nucleic acid into the host. It is anticipated that this lysozyme has a similar function to the lysozyme of other bacteriophages such as T4.  Determining the high resolution structure of the protein will allow us to later determine its location within the intact phage.            The gene of interest has been cloned into E. coli by PCR amplification and ligation into the PET30a vector. The protein expressed to high levels after IPTG induction. The His-tagged recombinant protein was then purified to homogeneity using a Ni-NTA column followed by size exclusion chromatography on a GE superose 6 column. The purified protein is currently undergoing crystallization trials. Thereafter, crystals of the protein will be used to determine the X-ray structure of the lysozyme.  The ultimate goal is to fit the putative lysozyme X-ray coordinates into the cryo-EM reconstruction of the bacteriophage SN.