Functional characterization of a naturally occurring trans-splicing intein from Synechococcus elongatus in a mammalian cell system.
We have cloned and characterized a naturally occurring split mini-DnaE intein capable of protein trans-splicing in the cyanobacterium Synechococcus elongatus (Sel DnaE intein). Sel DnaE intein is homologous to Synechocystissp. 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.
Chen
Linjie
L
Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang 310058, China.
Zhang
Yaping
Y
Li
Guo
G
Huang
Haishan
H
Zhou
Naiming
N
D000595:Amino Acid Sequence; D002460:Cell Line; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000037:antagonists & inhibitors / Q000378:metabolism; D023581:HIV Fusion Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D047668:Inteins; D008969:Molecular Sequence Data; D046937:Nostoc / Q000201:enzymology; D019154:Protein Splicing; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046940:Synechococcus / Q000201:enzymology*
D015699:HIV Envelope Protein gp120; D023581:HIV Fusion Inhibitors; D011993:Recombinant Fusion Proteins; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III