Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

In vitro expression and analysis of secreted fowlpox virus CC chemokine-like proteins Fpv060, Fpv061, Fpv116 and Fpv121

In vitro expression and analysis of secreted fowlpox virus CC chemokine-like proteins Fpv060,... Summary.The four CC chemokine-like proteins (Fpv060, Fpv061, Fpv116 and Fpv121) of fowlpox virus (FWPV) were over-expressed as His-tagged versions from a T7 promoter/EMCV IRES construct in vitro, by coupled transcription/translation, or in cell culture, by co-infection with two recombinant FWPVs (one expressing the chemokine-like protein and one expressing T7 RNA polymerase). All, except Fpv116, appeared to be glycosylated in the presence of microsomal membranes in vitro. In culture, all were secreted (even though secretion of Fpv061 was not predicted). Secreted forms of Fpv060 and Fpv121 were the most abundant forms of those two proteins. Glycosidase analysis of cellular and secreted forms confirmed that Fpv060, Fpv061 and Fpv121 were N-glycosylated and that the most abundant, cellular form of Fpv061 had been glycosylated but remained Endo H-sensitive (retained in the endoplasmic reticulum or Golgi). N-terminal sequence analysis of His-tagged Fpv060 and Fpv121 showed that they were processed at the predicted signal cleavage sites. Fpv060- and Fpv061-specific antipeptide sera allowed confirmation that the expression, processing and secretion of the native proteins were as determined for the His-tagged proteins. Isolation of knock-out mutants showed that all four proteins were non-essential for replication in tissue culture. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Virology Springer Journals

In vitro expression and analysis of secreted fowlpox virus CC chemokine-like proteins Fpv060, Fpv061, Fpv116 and Fpv121

Loading next page...
 
/lp/springer_journal/in-vitro-expression-and-analysis-of-secreted-fowlpox-virus-cc-DPYGYLmK1Q

References (29)

Publisher
Springer Journals
Copyright
Copyright © Springer-Verlag/Wien 2005
Subject
Biomedicine; Medical Microbiology; Infectious Diseases; Virology
ISSN
0304-8608
eISSN
1432-8798
DOI
10.1007/s00705-005-0560-7
pmid
15931460
Publisher site
See Article on Publisher Site

Abstract

Summary.The four CC chemokine-like proteins (Fpv060, Fpv061, Fpv116 and Fpv121) of fowlpox virus (FWPV) were over-expressed as His-tagged versions from a T7 promoter/EMCV IRES construct in vitro, by coupled transcription/translation, or in cell culture, by co-infection with two recombinant FWPVs (one expressing the chemokine-like protein and one expressing T7 RNA polymerase). All, except Fpv116, appeared to be glycosylated in the presence of microsomal membranes in vitro. In culture, all were secreted (even though secretion of Fpv061 was not predicted). Secreted forms of Fpv060 and Fpv121 were the most abundant forms of those two proteins. Glycosidase analysis of cellular and secreted forms confirmed that Fpv060, Fpv061 and Fpv121 were N-glycosylated and that the most abundant, cellular form of Fpv061 had been glycosylated but remained Endo H-sensitive (retained in the endoplasmic reticulum or Golgi). N-terminal sequence analysis of His-tagged Fpv060 and Fpv121 showed that they were processed at the predicted signal cleavage sites. Fpv060- and Fpv061-specific antipeptide sera allowed confirmation that the expression, processing and secretion of the native proteins were as determined for the His-tagged proteins. Isolation of knock-out mutants showed that all four proteins were non-essential for replication in tissue culture.

Journal

Archives of VirologySpringer Journals

Published: Sep 1, 2005

Keywords: Endoplasmic Reticulum; Cleavage Site; Native Protein; Microsomal Membrane; Secrete Form

There are no references for this article.