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Title: Development of E. coli strains with novel Tat-based export systems for therapeutic protein production
Author: Frain, Kelly May
ISNI:       0000 0004 7659 4845
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2018
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The Twin Arginine Translocase is a protein transport system in the inner membrane of both Gram-negative (E. coli) and Gram-positive (B. subtilis) bacteria. The translocase is unique as it exports fully folded substrates to the periplasm, moreover, it preferentially exports correctly folded proteins. Less information is known about the minimal Gram-positive translocases, TatAdCd and TatAyAy, but their 'proofreading' abilities and structure, respectively, are investigated alongside the usefulness of Tat in the biopharmaceutical industry. Initial studies (chapter 3) addressed which pharmaceutically recombinant proteins TatAdCd could export with a Tat specific signal peptide, TorA, to the periplasm. Surprisingly, the translocase only exported two scFv's, but by testing export of mutant scFv's I was able to determine, for the first time, the level of proofreading selectively by a Gram-positive Tat system. Given this knowledge, 8 scFv constructs in varying orientations from MedImmune were tested exploiting TatAdCd, Tat express and CyDisCo (chapter 4). I endeavored to optimize conditions for their expression and export through means of alternative medias and the fermentation process. Finally, given that Gram-positive Tat has unusual specificities and a minimal translocase, I used novel approaches to purify and characterise TatAyCy with SMA and CryoEM techniques (Chapter 5). The detailed insight provided in this study could help towards understanding the elusive translocation mechanism of Tat.
Supervisor: Robinson, Colin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available