Bacterial Expression of Human beta-Urogastrone from a Cloned Synthetic Gene

Fotheringham, Ian Graham (1986) Bacterial Expression of Human beta-Urogastrone from a Cloned Synthetic Gene. PhD thesis, University of Glasgow.

Abstract

This work describes the design, cloning and expression of a synthetic gene encoding a human peptide hormone, beta-urogastrone-epidermal growth factor. This is a 53 amino-acid polypeptide incorporating two separate biological activities: i) It inhibits the secretion of gastric HCl and ii) It stimulates growth of epidermal cells both in vivo and in cell culture. Preliminary studies using beta-urogastrone isolated from human urine have indicated that the peptide may have potential medical applications in the treatment of gastric ulcers. However its isolation and purification from this source is extremely inefficient and time consuming. Bacterial expression was therefore investigated as a viable alternative source of the peptide. In addition, total gene synthesis was examined as a suitable alternative to the isolation of the gene from natural sources. Various theoretical and practical concepts of gene design are discussed. The preliminary expression of beta-urogastrone in E. coli was carried out, using regulatory regions derived from the host tryptophan (trp), biosynthetic operon. Subsequently a series of vectors were designed to enable expression in an alternative host, B. subtilis. The expression system was incorporated on a dual-replicon plasmid allowing selectable propagation and a study of expression in both B. subtilis and in E. coli. Transcription was initiated from an early promoter derived from a B. subtilis phage and fully characterised here. Translation was achieved using synthetic ribosome binding sites. Subsequently, the use of site-directed mutagenesis permitted the development of this system to enable the future expression of additional genes.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Genetics, Endocrinology
• Date of Award: 1986
• Depositing User: Enlighten Team
• Unique ID: glathesis:1986-76586
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 14:06
• Last Modified: 19 Nov 2019 14:06
• URI: https://theses.gla.ac.uk/id/eprint/76586