Orthogonal dual-modification of proteins for the engineering of multivalent protein scaffolds.

Michaela Mühlberg,Nediljko Budisa,Michael G Hoesl,Christian Kuehne,Jens Dernedde,Christian P R Hackenberger

doi:10.3762/bjoc.11.88

Abstract

To add new tools to the repertoire of protein-based multivalent scaffold design, we have developed a novel dual-labeling strategy for proteins that combines residue-specific incorporation of unnatural amino acids with chemical oxidative aldehyde formation at the N-terminus of a protein. Our approach relies on the selective introduction of two different functional moieties in a protein by mutually orthogonal copper-catalyzed azide–alkyne cycloaddition (CuAAC) and oxime ligation. This method was applied to the conjugation of biotin and β-linked galactose residues to yield an enzymatically active thermophilic lipase, which revealed specific binding to Erythrina cristagalli lectin by SPR binding studies.

Highlights

The chemical modification of proteins has been developed to a core discipline in chemical biology with diverse applications in all areas of the life sciences, including pharmacology, biophysics, biotechnology and cell biology [1,2,3,4]
To add new tools to the repertoire of protein-based multivalent scaffold design, we have developed a novel dual-labeling strategy for proteins that combines residue-specific incorporation of unnatural amino acids with chemical oxidative aldehyde formation at the N-terminus of a protein
In a proof-of-principle study to engineer multivalent glycoprotein conjugates, we have used the incorporation of non-canonical amino acids (NCAA) [13] by supplementation based incorporation (SPI) [14,15,16,17] in auxotroph expression systems followed by the chemoselective Cu-catalyzed azide–alkyne cycloaddition (CuAAC) to attach carbohydrate ligands to the protein barstar [18]

Summary

Introduction

The chemical modification of proteins has been developed to a core discipline in chemical biology with diverse applications in all areas of the life sciences, including pharmacology, biophysics, biotechnology and cell biology [1,2,3,4]. In addition to the use of chemical labeling methods to study structure and function of proteins in vitro and in vivo, chemoselective conjugation techniques are used to functionalize artificial protein scaffolds, such as viral capsids [5,6,7]. Such templates have selfassembled hierarchical structures that allow the generation of nanostructured scaffolds with precisely defined dimensions and Beilstein J. We have recently contributed to this field using globular proteins as multivalent scaffolds for the structurally-defined presentation of ligands. In a proof-of-principle study to engineer multivalent glycoprotein conjugates, we have used the incorporation of non-canonical amino acids (NCAA) [13] by supplementation based incorporation (SPI) [14,15,16,17] in auxotroph expression systems followed by the chemoselective Cu-catalyzed azide–alkyne cycloaddition (CuAAC) to attach carbohydrate ligands to the protein barstar [18]

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Results

Conclusion