Journal
ANALYTICAL BIOCHEMISTRY
Volume 407, Issue 2, Pages 180-187Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2010.08.018
Keywords
DnaE intein; Protein trans-splicing; Protein reconstitution; Cell-cell fusion; HIV
Funding
- Science and Technology Department of Zhejiang Province [2007C23031]
- National Natural Science Foundation of China [30670425]
- Ministry of Science and Technology [2008AA02Z138]
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We have cloned and characterized a naturally occurring split mini-DnaE intein capable of protein trans-splicing in the cyanobacterium Synechococcus elongatus (Se! DnaE intein). Se! DnaE intein is homologous to Synechocystis sp. PCC6803 (Ssp) DnaE intein and Nostoc punctiforme (Npu) DnaE intein, with a protein sequence identity of 60% for the N-terminal part of intein and 61% for the C-terminal part of intein. Our results demonstrate that the split reporters, split Renilla luciferase (Rluc) and enhanced green fluorescent protein (EGFP), can be reconstituted via Sel DnaE intein-mediated trans-splicing in mammalian cells. Based on Sel DnaE intein-mediated reconstitution of split Rluc, a human immunodeficiency virus (HIV) entry-mimicking cell-cell fusion assay was developed and validated as a useful assay for screening and pharmacologically characterizing potential HIV entry-targeting inhibitors. (C) 2010 Elsevier Inc. All rights reserved.
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