Cloning, expression, and purification of the major tail protein in bacteriophage lambda

Katie Schwarz¹, William Wikoff², Biology Department, Washington University, St. Louis, MO^1, Department of Biochemistry, Washington University School of Medicine²

Virus tail assembly is an important problem in the field of virology, yet little is known about the protein that comprises the majority of the tail. Though it is known that the tubular section of the lambda bacteriophage tail is composed of 32 subunits of hexamer rings of gene product V (gpV), the protein’s tertiary structure has not been determined. The long term objective of my project is to crystallize gpV so that the protein’s structure can be resolved.

Gene V was cloned from bacteriophage lambda genomic DNA into the expression vector pet21a at the restriction sites Nde1 and EcoR1, and expressed in E. coli. The clone was checked for mutations by DNA sequencing. The results matched the wild type lambda gene V sequence. The protein, which expressed at high levels, roughly 30% to 50% of total protein, was partially purified and characterized using combinations of ammonium sulfate precipitations and gel filtration chromatography.

The results indicate that gpV forms a variety of aggregates in solutions with low ionic strength and neutral pH. Further experiments will be performed to determine how aggregation can be prevented or controlled so that crystallization experiments can be designed.