Polyionic fusion peptides function as specific dimerization motifs.
The de novo design of a molecular adapter for directed association and covalent linkage of two polypeptides is presented. Using peptides containing charged amino acid residues and an additional cysteine residue (AlaCysLys(8) and AlaCysGlu(8)) we demonstrate that the electrostatic interaction promotes the association of two synthetic peptides and, subsequently, disulfide bond formation. The reaction depends on both the redox potential and on the ionic strength of the buffer. Varying the redox potential, the interaction of the peptides was quantified by a Delta G(0') of 6.6 +/- 0.2 kcal/mol. Heterodimerization of the peptides is highly specific, a competition of association by other cysteine containing compounds could not be observed. Two proteins comprising cysteine-containing polyionic fusion peptides, a modified Fab fragment and an alpha-glucosidase fusion, could be specifically conjugated by directed association and subsequent disulfide bond formation. Both proteins retain their functional characteristics within the bifunctional conjugate: enzymatic activity of the alpha-glucosidase and antigen-binding capacity of the Fab fragment are equivalent to the non-conjugated components.
Richter
S A
SA
Institut für Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle, Germany.
Stubenrauch
K
K
Lilie
H
H
Rudolph
R
R
D000818:Animals; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019281:Dimerization; D004926:Escherichia coli; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007477:Ions; D051379:Mice; D010084:Oxidation-Reduction; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae; D000520:alpha-Glucosidases / Q000235:genetics / Q000378:metabolism
D007140:Immunoglobulin Fab Fragments; D007477:Ions; D011506:Proteins; D011993:Recombinant Fusion Proteins; D000520:alpha-Glucosidases