title: Expression, purification, crystallization and preliminary X-ray diffraction analysis of a lactococcal bacteriophage small terminase subunit.
abstract: Terminases are enzymes that are required for the insertion of a single viral genome into the interior of a viral procapsid by a process referred to as 'encapsulation or packaging'. Many double-stranded DNA viruses such as bacteriophages T3, T4, T7, λ and SPP1, as well as herpes viruses, utilize terminase enzymes for this purpose. All the terminase enzymes described to date require two subunits, a small subunit referred to as TerS and a large subunit referred to as TerL, for in vivo activity. The TerS and TerL subunits interact with each other to form a functional hetero-oligomeric enzyme complex; however the stoichiometry and oligomeric state have not been determined. We have cloned, expressed and purified recombinant small terminase TerS from a 936 lactococcal bacteriophage strain ASCC454, initially isolated from a dairy factory. The terminase was crystallized using a combination of nanolitre sitting drops and vapour diffusion using sodium malonate as the precipitant, and crystallization optimized using standard vapour-diffusion hanging drops set up in the presence of a nitrogen atmosphere. The crystals belong to the P2 space group, with unit-cell parameters a=73.93, b=158.48, c=74.23 Å, and diffract to 2.42 Å resolution using synchrotron radiation. A self-rotation function calculation revealed that the terminase oligomerizes into an octamer in the asymmetric unit, although size-exclusion chromatography suggests that it is possible for it to form an oligomer of up to 13 subunits.
authors.affiliation: Materials Science and Engineering, CSIRO, 343 Royal Parade, Parkville, Victoria 3052, Australia.
authors.forename: Bin
authors.initials: B
authors.lastname: Ren
authors.forename: Tam M
authors.initials: TM
authors.lastname: Pham
authors.forename: Regina
authors.initials: R
authors.lastname: Surjadi
authors.forename: Christine P
authors.initials: CP
authors.lastname: Robinson
authors.forename: Thien-Kim
authors.initials: TK
authors.lastname: Le
authors.forename: P Scott
authors.initials: PS
authors.lastname: Chandry
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Peat
authors.forename: William J
authors.initials: WJ
authors.lastname: McKinstry
mesh_terms: D001435:Bacteriophages / Q000737:chemistry* / Q000201:enzymology; D003460:Crystallization; D018360:Crystallography, X-Ray; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D017045:Lactococcus / Q000821:virology; D055503:Protein Multimerization; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; D014764:Viral Proteins; D004706:Endodeoxyribonucleases; C030894:terminase
keywords: DNA-binding protein; Siphoviridae bacteriophage; small terminase subunit