Tight transcriptional regulation of foreign genes in insect cells using an ecdysone receptor-based inducible system

Xiaojiang Dai, Leslie G. Willis, Subba R. Palli, David A. Theilmann

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

The use of insect cells has been highly successful for the expression of foreign proteins from baculoviruses or plasmid vectors. Here, we describe a tight transcriptional regulation of foreign genes in insect cells using an ecdysone receptor-based inducible system. The system includes the DEF domains of the spruce budworm (Choristoneura fumiferana) EcR (CfEcR) fused to the Saccharomyces cerevisiae GAL4 DNA-binding domain and the EF domains of mammalian Mus musculus retinoid X receptor (MmRXR) fused to the acidic activation domains (AADs) of the baculovirus transactivators IE1 and IE0. Using a GAL4 response element in reporter constructs, both transient and stable expression in insect lepidopteran cells showed that the chimeric MmRXR and CfEcR only activated the reporter genes in the presence of inducer; no gene expression was detectable in the absence of inducer. Characterization of heterogenous activation domains in insect cells showed that the AADs from Autographa californica multiple nucleopolyhedrovirus (MNPV) IE1 and Orgyia pseudotsugata MNPV IE0 consistently exhibited higher inducible levels than the archetype AAD from herpesvirus VP16 in insect cells. To confirm the tight regulation of this system the highly toxic protein, diphtheria toxin (DT), was used. In the absence of an inducer no cytotoxic effect was observed in insect cells that had been transiently transformed with DT expressing plasmids. This system will therefore be a very useful tool for biotechnology applications expressing highly toxic proteins in insect cells and for studying the functional genomics of insects and microorganisms that infect them.

Original languageEnglish
Pages (from-to)236-245
Number of pages10
JournalProtein Expression and Purification
Volume42
Issue number2
DOIs
StatePublished - Aug 2005

Bibliographical note

Funding Information:
We thank Dr. David Potter (Rheogene Co.) for supplying plasmid 6×GALRE-TI-DT and Guus Bakkeren (Pacific Agri-Food Research Centre) for pCM54. The present study was supported by Agriculture and Agri-Food Canada and Rheogene Co. through the Matching Investment Initiative.

Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.

Keywords

  • Baculovirus
  • Ecdysone receptor
  • Inducible expression
  • Stably transformed insect cells
  • VP16

ASJC Scopus subject areas

  • Biotechnology

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