Insect expression systems : improving intracellular and heterologous gene expression
type: CreativeWork
description: Expression systems (ES) in a wide variety of biological systems are used to provide foreign protein. Protein production in mammalian cells is a labour-intensive and expensive process and insect cells have been used as cheaper alternatives. Insect ES are based on two types of vector; viral- and plasmid-based. The baculovirus ES (BES) offers high-level transient production of intracellular proteins. Yields of secreted and membrane-targeted proteins have until recently been relatively low, however, the development of a novel vector, lacking chitinase, has overcome these limitations. Plasmid-based vectors can be used for transient and stable expression in insect cells but most utilise the relatively weak Autographa californica multiple nucleopolyhedrovirus (AcMNPV) immediate early-1 (ie-1) promoter, producing low-levels of intracellular recombinant protein. More recent vectors have used stronger promoters, however, compared with mammalian stable ES these are limited in use and most notable is the lack of an efficient inducible ES in insect cells. The work in this thesis describes methods, with which intracellular levels of foreign protein may be increased, while also developing the grounding work for the development of a fully functional insect tetracycline regulatory system. To improve stable and regulated expression of heterologous genes in insect cells, work was undertaken to compare and characterise the transcriptional activity of a range of promoters successfully used in insect and mammalian systems. Of the promoters tested, Orgyia pseudotsugata MNPV (Op) ie-2 and Bombyx morl actin 3 (BmA3+E) were found to be transcriptionally stronger than AcMNPV ie-1. Drosophila melanogaster metallothionein and actin 5.1 did not produce any detectable activity, and the Cytomegalovirus (CMV) ie promoter, although active, was weaker than AcMNPV ;e-1. Attempts to develop a new inducible ES for use in stable cell lines was based on modifying a mammalian tetracyclineinducible ES. The …
abstract: Expression systems (ES) in a wide variety of biological systems are used to provide foreign protein. Protein production in mammalian cells is a labour-intensive and expensive process and insect cells have been used as cheaper alternatives. Insect ES are based on two types of vector; viral- and plasmid-based. The baculovirus ES (BES) offers high-level transient production of intracellular proteins. Yields of secreted and membrane-targeted proteins have until recently been relatively low, however, the development of a novel vector, lacking chitinase, has overcome these limitations. Plasmid-based vectors can be used for transient and stable expression in insect cells but most utilise the relatively weak Autographa californica multiple nucleopolyhedrovirus (AcMNPV) immediate early-1 (ie-1) promoter, producing low-levels of intracellular recombinant protein. More recent vectors have used stronger promoters, however, compared with mammalian stable ES these are limited in use and most notable is the lack of an efficient inducible ES in insect cells. The work in this thesis describes methods, with which intracellular levels of foreign protein may be increased, while also developing the grounding work for the development of a fully functional insect tetracycline regulatory system. To improve stable and regulated expression of heterologous genes in insect cells, work was undertaken to compare and characterise the transcriptional activity of a range of promoters successfully used in insect and mammalian systems. Of the promoters tested, Orgyia pseudotsugata MNPV (Op) ie-2 and Bombyx morl actin 3 (BmA3+E) were found to be transcriptionally stronger than AcMNPV ie-1. Drosophila melanogaster metallothionein and actin 5.1 did not produce any detectable activity, and the Cytomegalovirus (CMV) ie promoter, although active, was weaker than AcMNPV ;e-1. Attempts to develop a new inducible ES for use in stable cell lines was based on modifying a mammalian tetracyclineinducible ES. The …
author: Richards, Kevin S.
language: en
date published: 2004
date modified: 2026-07-24T03:43:52Z
provider: Oxford Brookes University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Oxford Brookes University

PropertyValue
property ID: DOI
value: 10.24384/ct4t-1848