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Protein Science (2004), 13:2458-2469. Published by Cold Spring Harbor Laboratory Press. Copyright © 2004 The Protein Society
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A directed evolution strategy for optimized export of recombinant proteins reveals critical determinants for preprotein discharge

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

Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion, SY23 3DD, United Kingdom

(RECEIVED February 16, 2004; FINAL REVISION June 9, 2004; ACCEPTED June 9, 2004)

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.

Keywords: protein secretion; protein translocation; signal sequence; directed evolution; cytochrome b5; bioenergetics

Abbreviations: E, Energization state • m-CCCP, m-chloro carbonyl cyanide phenylhydrazone • PAGE, polyacrylamide gel electrophoresis • P, protonophoric force • SDS, sodium dodecyl sulphate • RH123, Rhodamine 123.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04697304.

Reprint requests to: Mustak A. Kaderbhai, Institute of Biological Sciences, Cledwyn Building, University of Wales, Aberystwyth, Ceredigion, SY23 3DD, UK; e-mail: mak{at}aber.ac.uk; fax: +44-1970-622294.


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