title: Purification of full-length VP22 from cells infected with HSV-1: A two-pronged approach for the solubilization and purification of viral proteins for use in biochemical studies.
abstract: VP22, encoded by the U(L)49 gene, is one of the most abundant proteins of the herpes simplex virus type 1 (HSV-1) tegument and has been shown to be important for virus replication and spread. However, the exact role(s) played by VP22 in the HSV-1 replication cycle have yet to be delineated. The lack of a procedure to purify full-length VP22 has limited molecular studies on VP22 function. A procedure was developed for the purification of soluble, full-length VP22 from cells infected with HSV-1. A recombinant virus encoding His-tagged VP22 was generated and found to express VP22 at levels comparable to the wild type virus upon infection of Vero cells. By experimenting with a wide variety of cell lysis buffer conditions, several buffers that promote the solubility of full-length VP22 were identified. Buffers that gave the highest levels of solubility were then used in immobilized metal ion affinity chromatography experiments to identify conditions that provided the greatest level of VP22 binding and recovery from cobalt and nickel affinity resins. Using this strategy soluble, full-length VP22 was purified from cells infected with HSV-1.
authors.affiliation: Department of Biological Sciences, University of Alabama, 300 Hackberry Lane, Tuscaloosa, AL 35487, USA.
authors.forename: Ebony J
authors.initials: EJ
authors.lastname: Dewberry
authors.forename: Eric
authors.initials: E
authors.lastname: Dunkerley
authors.forename: Carol
authors.initials: C
authors.lastname: Duffy
mesh_terms: D000818:Animals; D002021:Buffers; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018259:Herpesvirus 1, Human / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D012995:Solubility; D014709:Vero Cells; D015678:Viral Structural Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D002021:Buffers; D011993:Recombinant Fusion Proteins; D015678:Viral Structural Proteins; C073413:herpes simplex virus type 1 protein VP22