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|Title:||High level gene expression in E.coli: Effects of point mutations within the coding sequence.|
|Authors:||Wright, Edwina M.|
|Presented at:||University of Leicester|
|Abstract:||E.coli vectors having thermo-inducible copy number have been described by Yarranton et al. (1984). This thesis describes modification of such vectors to improve both their segregational stability and their host strain versatility. The modified vector system was initially used to demonstrate efficient, controllable expression of two inserted genes, one prokaryotic and one eukaryotic. This stable and efficient vector system was subsequently used to overexpress the eukaryotic gene TIMP (tissue inhibitor of metalloproteinases). Examination of expression levels of the recombinant protein revealed that point mutations within 5' end of the structural gene caused up to five-fold variation in the levels accumulated by the cells. Not all of the base substitutions caused changes in the amino acid sequence of the protein. With the aim of learning more about some of the factors influencing gene expression and because such accumulation differences are important in the large scale production of foreign proteins in E. coli, possible explanations for this phenomenon were sought. There were no detectable differences between copy numbers of the vectors carrying the mutant genes, or in either the levels or half lives of TIMP-specific mRNA in cells carrying each vector. The differences in protein accumulation were shown to be primarily due to variations in the rates of synthesis but differences in degradation rates were also detected. The differences in protein synthesis rates occur at the level of translation, however, in a computer search no significant alterations in mRNA secondary structure which could affect translational initiation were found. In vitro studies are described, in which attempts were made to demonstrate that translation rate differences are caused by variations in affinities of the mRNAs for the ribosomes.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Genetics|
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