GB Tags: Small Covalent Peptide Tags Based on Protein Fragment Reconstitution

Developing peptide tags that can bind target proteins covalently under mild conditions is of great importance for a myriad of applications, ranging from chemical biology to biotechnology. Here we report the development of a small covalent peptide tag system, termed as GB tags, that can covalently label the target protein with high specificity and high yield under oxidizing conditions. The GB tags consist of a pair of short peptides, GN and GC (GN contains 45 residues and GC contains 19 residues). GN and GC, which are split from a parent protein GB1, can undergo protein fragment reconstitution to reconstitute the folded structure of the parent protein spontaneously. The engineered cysteines in GN and GC can readily form a disulfide bond oxidized by air oxygen after protein reconstitution. Using thermally stable variants of GB1, we identified two pairs of GB tags that display improved thermodynamic stability and binding affinity. They can serve as efficient covalent peptide tags for various applications, including specific labeling of mammalian cell surface receptors. We anticipate that these new GB tags will find applications in biochemical labeling as well as biomaterials, such as protein hydrogels.