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Protein expression and purification2011; 80(2); 274-282; doi: 10.1016/j.pep.2011.08.002

Factors affecting recombinant Western equine encephalitis virus glycoprotein production in the baculovirus system.

Abstract: In an effort to produce processed, soluble Western equine encephalitis virus (WEEV) glycoproteins for subunit therapeutic vaccine studies, we isolated twelve recombinant baculoviruses designed to express four different WEEV glycoprotein constructs under the transcriptional control of three temporally distinct baculovirus promoters. The WEEV glycoprotein constructs encoded full-length E1, the E1 ectodomain, an E26KE1 polyprotein precursor, and an artificial, secretable E2E1 chimera. The three different promoters induced gene expression during the immediate early (ie1), late (p6.9), and very late (polh) phases of baculovirus infection. Protein expression studies showed that the nature of the WEEV construct and the timing of expression both influenced the quantity and quality of recombinant glycoprotein produced. The full-length E1 product was insoluble, irrespective of the timing of expression. Each of the other three constructs yielded soluble products and, in these cases, the timing of expression was important, as higher protein processing efficiencies were generally obtained at earlier times of infection. However, immediate early expression did not yield detectable levels of every WEEV product, and expression during the late (p6.9) or very late (polh) phases of infection provided equal or higher amounts of processed, soluble product. Thus, while earlier foreign gene expression can provide higher recombinant glycoprotein processing efficiencies in the baculovirus system, in the case of the WEEV glycoproteins, earlier expression did not provide larger amounts of high quality, soluble recombinant glycoprotein product.
Copyright © 2011 Elsevier Inc. All rights reserved.
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Publication Date: 2011-08-16 PubMed ID: 21864686PubMed Central: PMC3196263DOI: 10.1016/j.pep.2011.08.002Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article focuses on the production of soluble Western equine encephalitis virus (WEEV) glycoproteins using the baculovirus system and the factors that affect the quantity and the quality of this production. It presents the outcomes of various constructs and promoters and how they influence the process.

Experiment Design:

  • The researchers created twelve recombinant baculoviruses. These are made to express four different WEEV glycoprotein constructs.
  • The purpose of these constructs is to express the full-length E1, the E1 ectodomain, an E26KE1 polyprotein precursor, and an artificial, secretable E2E1 chimera.
  • The constructs come under the transcriptional control of three different baculovirus promoters, which induce gene expression during the immediate early (ie1), late (p6.9), and very late (polh) phases of baculovirus infection.

Results:

  • The observed results showed that the type of WEEV construct and the timing of expression significantly influenced the amount and quality of the recombinant glycoprotein produced.
  • The full-length E1 product resulted in an insoluble state, regardless of when it was expressed.
  • Each of the other three constructs produced soluble products at different times of expression with higher protein processing efficiencies usually achieved at earlier times of infection.
  • The immediate early expression did not yield detectable levels for all WEEV products.
  • Expression during the late (p6.9) or very late (polh) phases of infection provided equal or larger amounts of processed, soluble product.

Conclusion:

  • Thus, the study concludes that while early foreign gene expression can result in higher recombinant glycoprotein processing efficiencies in the baculovirus system, in the case of the WEEV glycoproteins, earlier expression did not provide larger amounts of high-quality, soluble recombinant glycoprotein product.
  • This shows the complex relationship between the timing of gene expression and the type of construct used in the production of recombinant glycoproteins for therapeutic vaccine studies.

Cite This Article

APA
Toth AM, Geisler C, Aumiller JJ, Jarvis DL. (2011). Factors affecting recombinant Western equine encephalitis virus glycoprotein production in the baculovirus system. Protein Expr Purif, 80(2), 274-282. https://doi.org/10.1016/j.pep.2011.08.002

Publication

Protein expression and purification
ISSN: 1096-0279
NlmUniqueID: 9101496
Country: United States
Language: English
Volume: 80
Issue: 2
Pages: 274-282

Researcher Affiliations

Toth, Ann M
  • Department of Molecular Biology, University of Wyoming, Laramie, WY 82071, USA.
Geisler, Christoph
    Aumiller, Jared J
      Jarvis, Donald L

        MeSH Terms

        • Animals
        • Baculoviridae / genetics
        • Baculoviridae / isolation & purification
        • Baculoviridae / metabolism
        • Baculoviridae / pathogenicity
        • Blotting, Western
        • Cell Line
        • Cloning, Molecular
        • Encephalitis Virus, Western Equine / chemistry
        • Encephalitis Virus, Western Equine / genetics
        • Gene Expression Regulation, Viral
        • Genes, Viral
        • Genetic Vectors / genetics
        • Genetic Vectors / metabolism
        • Glycoproteins / genetics
        • Glycoproteins / isolation & purification
        • Glycoproteins / metabolism
        • Glycosylation
        • Promoter Regions, Genetic
        • Recombinant Proteins / genetics
        • Recombinant Proteins / isolation & purification
        • Recombinant Proteins / metabolism
        • Solubility
        • Spodoptera
        • Time Factors
        • Transcription, Genetic
        • Viral Envelope Proteins / genetics
        • Viral Envelope Proteins / isolation & purification
        • Viral Envelope Proteins / metabolism

        Grant Funding

        • U54 AI065357-06 / NIAID NIH HHS
        • U54 AI-065357 / NIAID NIH HHS
        • P41 RR005351 / NCRR NIH HHS
        • P41 RR005351-19 / NCRR NIH HHS
        • U54 AI065357 / NIAID NIH HHS

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