A directed evolution strategy for optimized export of recombinant proteins reveals critical determinants for preprotein discharge

Mustak A. Kaderbhai, Hazel M. Davey, Naheed N. Kaderbhai

Allbwn ymchwil: Cyfraniad at gyfnodolynErthygladolygiad gan gymheiriaid

18 Dyfyniadau(SciVal)

Crynodeb

A directed evolutionary approach is described that searches short, random peptide sequences for appendage at the secretory signal peptide–mature protein junction to seek ideal algorithms for both efficient and hyper export of recombinant proteins to the periplasm of Escherichia coli. The strategy employs simple, visual detection of positive clones using a PINK expression system that faithfully reports on export status of a mammalian hemoprotein in E. coli. With-in 'sequence spaces' ranging from 1 to 13 residues, a significant but highly variable secretory fitness was scored such that the rate of secretion reciprocally correlated with the membrane-associated precursor pool of the evolved exportable hemoproteins. Three clusters of hyper, median, and hypo exporters were isolated. These had corresponding net charges of –1, 0, and +1 within the evolved sequence space, which in turn clearly correlated with the prevailing magnitude and polarity of the membrane energization states. The findings suggest that both the nature of the charged residue and the proximal sequence in the early mature region are the crucial determinants of the protonophore-dependent electrophoretic discharge of the precursor across the inner membrane of E. coli. We conclude that the directed evolutionary approach will find ready application in engineering recombinant proteins for their efficient secretion via the sec export pathway in E. coli.
Iaith wreiddiolSaesneg
Tudalennau (o-i)2458-2469
Nifer y tudalennau12
CyfnodolynScience
Cyfrol13
Rhif cyhoeddi9
Dynodwyr Gwrthrych Digidol (DOIs)
StatwsCyhoeddwyd - 01 Medi 2004

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