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báo cáo hóa học:" Development of an in vitro cleavage assay system to examine vaccinia virus I7L cysteine proteinase activity"

Tuyển tập các báo cáo nghiên cứu về hóa học được đăng trên tạp chí sinh học đề tài : Development of an in vitro cleavage assay system to examine vaccinia virus I7L cysteine proteinase activity | Virology Journal BioMed Central Research Open Access Development of an in vitro cleavage assay system to examine vaccinia virus I7L cysteine proteinase activity Chelsea M Byrd1 and Dennis E Hruby 1 2 Address 1Molecular and Cellular Biology Program Oregon State University 220 Nash Hall Corvallis Oregon 97331 USA and 2Siga Technologies 4575 SW Research Way Suite 230 Corvallis Oregon 97333 USA Email Chelsea M Byrd - cbyrd@sgph.com Dennis E Hruby - dhruby@sgph.com Corresponding author Published 16 August 2005 Received 21 April 2005 Accepted 16 August 2005 Virology Journal 2005 2 63 doi 10.1186 1743-422X-2-63 This article is available from http www.virologyj.cOm content 2 1 63 2005 Byrd and Hruby licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License http creativecommons.org licenses by 2.0 which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited. Abstract Through the use of transient expression assays and directed genetics the vaccinia virus VV I7L gene product has been implicated as the major maturational proteinase required for viral core protein cleavage to occur during virion assembly. To confirm this hypothesis and to enable a biochemical examination of the I7L cysteine proteinase an in vitro cleavage assay was developed. Using extracts of VV infected cells as the source of enzyme reaction conditions were developed which allowed accurate and efficient cleavage of exogenously added core protein precursors P4a P4b and P25K . The cleavage reaction proceeded in a time-dependent manner and was optimal when incubated at 25 C. I7L-mediated cleavage was not affected by selected inhibitors of metalloproteinases aspartic acid proteinases or serine proteinases EDTA pepstatin and PMSF respectively but was sensitive to several general cysteine proteinase inhibitors E-64 EST Iodoacetic acid and NEM as well as the I7L active site inhibitor .