Engineering of de novo pathways for oxidative protein folding in Escherichia coli
Lluis Masip (Departament d'Enginyeria Quimica, Universitat Rovira i Virgili)
Location: Sala de Graus, ETSEQ
Start time: Nov. 19, 2010,
12 p.m.
Abstract
In some proteins the formation of disulfide bonds, i.e. the covalent links joining the thiol groups in cysteine amino acids, is required in order to attain their biologically active three dimensional conformation. Many commercially important proteins contain disulfide bonds, and while the bacterium Escherichia coli is used extensively for recombinant protein manufacturing at an industrial scale, its ability to produce proteins containing multiple disulfide bonds is limited by biological constraints. This is because the cellular machinery that catalyzes the oxidation of cysteine residues resulting in the formation of disulfide bonds has evolved to cope with the relatively simple proteins of this organism which typically contain no more than 2-3 disulfides. The objective of my work has been to re-engineer the disulfide bond formation machinery of E. coli in order to improve our mechanistic understanding of disulfide bond formation and to provide additional tools for the expression of heterologous proteins with disulfide bonds in bacteria. Two examples of these re-engineered disulfide bond formation pathways will be discussed, the engineering of thioredoxin 1 to be a better periplasmic oxidant and the development of a less oxidizing periplasm.
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