The Poly-Histidine Tag H6 Mediates Structural and Functional Properties of Disintegrating, Protein-Releasing Inclusion Bodies.

Julieta María Sánchez,Esther Vazquez,Neus Ferrer-Miralles,Naroa Serna,Hèctor López-Laguna,Antonio Villaverde,José Vicente Carratalá,Eric Voltà-Durán,Ugutz Unzueta,Verónica Nolan,Alejandro Sánchez-Chardi

doi:10.3390/pharmaceutics14030602

Abstract

The coordination between histidine-rich peptides and divalent cations supports the formation of nano- and micro-scale protein biomaterials, including toxic and non-toxic functional amyloids, which can be adapted as drug delivery systems. Among them, inclusion bodies (IBs) formed in recombinant bacteria have shown promise as protein depots for time-sustained protein release. We have demonstrated here that the hexahistidine (H6) tag, fused to recombinant proteins, impacts both on the formation of bacterial IBs and on the conformation of the IB-forming protein, which shows a higher content of cross-beta intermolecular interactions in H6-tagged versions. Additionally, the addition of EDTA during the spontaneous disintegration of isolated IBs largely affects the protein leakage rate, again protein release being stimulated in His-tagged materials. This event depends on the number of His residues but irrespective of the location of the tag in the protein, as it occurs in either C-tagged or N-tagged proteins. The architectonic role of H6 in the formation of bacterial IBs, probably through coordination with divalent cations, offers an easy approach to manipulate protein leakage and to tailor the applicability of this material as a secretory amyloidal depot in different biomedical interfaces. In addition, the findings also offer a model to finely investigate, in a simple set-up, the mechanics of protein release from functional secretory amyloids.

Highlights

In the context of sustained drug delivery, a diversity of advanced materials for controlled release are under continuous development [1–4]
To explore the potential role of H6 in the formation of bacterial inclusion bodies (IBs) we produced in Escherichia coli the model protein VP1-GFP, as H6-tagged (VP1-GFP-H6) and untagged forms (Figure 1A)
The present study demonstrates that the addition of an H6 tag to a model fusion protein clearly influences the size of aggregates formed in recombinant bacteria, protein clearly influences the size of aggregates formed in recombinant bacteria, this impact is dissimilar depending on the tagged protein (Figures 1B,C and 4D)

Summary

Introduction

In the context of sustained drug delivery, a diversity of advanced materials for controlled release are under continuous development [1–4]. Most of them are based on mechanically stable, biologically inert networks or scaffolds that act as holders of the therapeutic molecule and from, which under physiological conditions, a leakage of this payload is allowed or promoted [5–7] This strategy is of particular interest in regenerative medicine that requires the time-prolonged local release of hormones and growth factors [8,9], and in other clinical conditions in which steady systemic drug levels are necessary to enhance efficacy, in contrast to undesired drug oscillations [10–14]. Due to the combination of structural and functional traits, protein drugs are especially suitable for this self-containing approach In this regard, the secretory granules from the human endocrine system are functional amyloids in which peptide hormones are self-stored through ion-assisted protein–protein interactions based on histidine residues [16–19]. This category of interactions between divalent cations such as Zn2+ and histidine residues, common in natural amyloidal structures [20–22], are fully reversible and ensure the physiological release of peptide hormones into the bloodstream

Methods

Results

Discussion

Conclusion