Split-GFP reassembly is an operationally simple in vivo technique used to identify and study interactions involving proteins and/or peptides. However, the instability of split-GFP fragments and their susceptibility to aggregation place limitations on the broader use of split-GFP reassembly. Supercharged proteins, including supercharged GFP, are variants with high theoretical negative or positive charge that are resistant to aggregation. We show that a split-superpositive GFP (split-spGFP) variant reassembles faster and more efficiently than previously reported split-sg100 GFP and split-folding-reporter GFP (split-frGFP) systems. In addition, interaction-dependent split-spGFP reassembly is efficient at physiological temperature. The increased efficiency and robustness of split-spGFP reassembly make this reporter system ideal for identifying and studying interactions involving proteins and/or peptides in vivo, and may be particularly useful for identifying or studying interactions involving proteins or peptides that are themselves susceptible to aggregation.

Protein engineering is an emerging discipline that dovetails modern molecular biology techniques with high-throughput screening, laboratory evolution technologies, and computational approaches to modify sequence, structure, and in some cases, function and properties of proteins. The ultimate goal is to develop new proteins with improved or designer functions for use in biotechnology, medicine and basic research. One way to engineer proteins is to change their solvent exposed regions through focused or random ‘protein resurfacing’. In this review we explain what protein resurfacing is, and discuss recent examples of how this strategy is used to generate proteins with altered or broadened recognition profiles, improved stability, solubility, expression, cell penetrating ability, and reduced immunogenicity. Additionally, we comment on how these properties can be further improved using chemical resurfacing approaches. Protein resurfacing will likely play an increasingly important role as more biologics enter clinical use, and we present some arguments to support this view.

Increased cellular levels of protein–protein interactions involving the ankyrin repeat oncoprotein gankyrin are directly linked to aberrant cellular events and numerous cancers. Inhibition of these protein–protein interactions is thus an attractive therapeutic strategy. However, the relatively featureless topology of gankyrin’s putative binding face and large surface areas involved in gankyrin-dependent protein–protein interactions present a dramatic challenge to small molecule discovery. The size, high folding energies, and well-defined surfaces present in many proteins overcome some of the challenges faced by small molecule discovery. We used split-superpositive Green Fluorescent Protein (split-spGFP) reassembly to screen a 5 × 109 library of resurfaced proteins that are shape complementary to the putative binding face of gankyrin and identified mutants that potently and selectively bind this oncoprotein in vitro and in living cells. Collectively, our findings represent the first synthetic proteins that bind gankyrin and may represent a general strategy for developing protein basic research tools and drug leads that bind disease-relevant ankyrin repeats.

Overexpression of the ankyrin repeat oncoprotein gankyrin is directly linked to the onset, proliferation and/or metastasis of many cancers. The role of gankyrin in multiple disease-relevant biochemical processes is profound. In addition to other cellular processes, gankyrin overexpression leads to decreased cellular levels of p53, through a complex that involves MDM2. Thus, inhibition of this interaction is an attractive strategy for modulating oncogenic phenotypes in gankyrin-overexpressing cells. However, the lack of well-defined hydrophobic small-molecule binding pockets on the putative ankyrin repeat binding face presents a challenge to traditional small-molecule drug discovery. In contrast, by virtue of their size and relatively high folding energies, synthetic gankyrin-binding proteins could, in principle, compete with physiologically relevant PPIs involving gankyrin. Previously, we showed that a shape-complementary protein scaffold can be resurfaced to bind gankyrin with moderate affinity (KD ~6 μM). Here, we used yeast display high-throughput screening, error-prone PCR, DNA shuffling, and protein engineering to optimize this complex. The best of proteins proteins bind gankyrin with excellent affinity (KD ~21 nM), selectively co-purify with gankyrin from a complex cellular milieu, modulate an interaction between gankyrin and a physiological binding partner (S6 ATPase), and suppress gankyrin/MDM2-dependent ubiquitination of p53.

Methods for the stabilization of well-defined helical peptide drugs and basic research tools have received considerable attention in the last decade. Here, we report the stable and functional display of an HIV gp41 C-peptide helix mimic on a GRAM-Like Ubiquitin-binding in EAP45 (GLUE) protein. C-peptide helix-grafted GLUE selectively binds the N-terminal helical region of gp41, a well-established HIV drug target, in a complex cellular environment. Additionally, the helix-grafted GLUE is folded in solution, stable in human serum, and soluble in aqueous solutions, and thus overcomes challenges faced by a multitude of peptide drugs, including those derived from HIV gp41 C-peptide.