A directed evolution strategy for optimized export of recombinant proteins reveals critical determinants for preprotein discharge.
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.
Kaderbhai
Mustak A
MA
Institute of Biological Sciences, Cledwyn Building, University of Wales, Aberystwyth, Ceredigion, SY23 3DD, UK. mak@aber.ac.uk
Davey
Hazel M
HM
Kaderbhai
Naheed N
NN
D000595:Amino Acid Sequence; D005075:Biological Evolution; D002462:Cell Membrane / Q000378:metabolism; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006420:Hemeproteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D019897:Periplasm / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism
D006420:Hemeproteins; D008565:Membrane Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D015786:Cytochromes b5; D012697:Serine Endopeptidases; C026515:type I signal peptidase