A Novel Method for the N-Terminal Modification of Native Proteins

Abstract

The formation of structurally defined bioconjugates of proteins hinges on their regioselective modification. Toward this goal a novel method is described here using the commercial IgA protease to attach a nonnatural peptidic moiety to the N-terminus of predisposed proteins by means of a kinetically controlled reverse proteolysis in water. The process requires an H-Ala-Pro N-terminal sequence and then furnishes a selectively modified conjugate under nondenaturing and nondestructive conditions in acceptable yield. The method lends itself to the N-terminal introduction of orthogonal moieties that may be elaborated further.

Branching Out of Single‐Molecule Fluorescence Spectroscopy: Challenges for Chemistry and Influence on Biology

“…To circumvent these problems, Lewinska et al described a novel method for the specific labeling of native proteins at a single, well‐defined position using the commercial IgA protease to attach a non‐natural peptidic moiety to the N terminus of the antibody. This natural peptidic moiety can be selectively modified 290. We are currently developing a new labeling strategy for antibodies which guaranties that practically all the antibodies present in the sample carry a single fluorophore (with only a small number of double‐labeled antibodies).…”

Phenyliodine(III) Diacetate (PIDA) Mediated Synthesis of Aromatic Azo Compounds through Oxidative Dehydrogenative Coupling of Anilines: Scope and Mechanism

“…The well‐known high specificity of this enzyme for its protein substrate limits its general suitability. Given the reversibility of enzymatic reactions, proteinases are also suitable for ligation reactions, as demonstrated for bacterial IgA protease13 and subtiligase 14. Nevertheless, natural proteases suffer from a major drawback: hydrolysis is generally highly favored over ligation, thus requiring careful kinetic or thermodynamic control of the reaction.…”

Neue Wege in der Einzelmolekül‐Fluoreszenzspektroskopie: Herausforderungen für die Chemie und Einfluss auf die Biologie

“…Diese Probleme umgingen Lewinska et al mit einer neuen Methode zur spezifischen Markierung nativer Proteine an einer einzigen Position. Mithilfe einer kommerziell erhältlichen IgA‐Protease hängten sie einen synthetischen Peptidabschnitt, der selektiv verändert werden kann, an das N‐terminale Ende der Antikörper an 290. Wir entwickeln zurzeit eine Markierungsstrategie für Antikörper, die garantiert, dass praktisch alle Antikörper in der Probe nur ein einziges Fluorophor tragen (bei einem geringen Anteil an Doppelmarkierungen).…”