The HaloTag: Improving Soluble Expression and Applications in Protein Functional Analysis

Exploration of protein function and interaction is critical for discovering links between genomics, proteomics, and disease state; yet the immense complexity of proteomics found in biological systems currently limit our investigational capacities. While affinity and autofluorescent tags are widely employed for protein analysis, these methods have limited success as they lack specificity and require multiple fusion tags and genetic constructs. As an alternative approach, the innovative HaloTag protein fusion platform allows for comprehensive analysis of protein function and interaction using a single genetic construct with multiple capabilities. This is accomplished using a simplified process, in which a variable HaloTag ligand binds rapidly to the HaloTag protein (usually linked to the protein of interest) with high affinity and specificity. In this review article, we examine all current applications of the HaloTag technology platform for biomedical applications such as the study of protein isolation and purification, protein function, protein-protein and protein-DNA interactions, biological assays, in vitro cellular imaging, and in vivo molecular imaging. In addition, novel uses of the HaloTag platform are briefly discussed with potential future applications.

show abstract

Section: Analyzing Protein–protein and Protein–dna Interactionsmentioning

confidence: 99%

HaloTag Technology: A Versatile Platform for Biomedical Applications

2015

View full text Add to dashboard Cite

show abstract

“…Although nanotechnology combined with bioscience has been developing rapidly with new bioconjugation approaches to be discovered, the guided nanoparticle delivery inside the cell remains a challenging task . Recently, a new modern approach, so‐called Halo Tag technology, has been designed to provide new options for rapid, site‐specific labeling of proteins in living cells and in vitro, and based on the efficient formation of a covalent bond between the Halo Tag protein and synthetic ligands ( Figure a) . Besides being used in biology for protein expression studies, this technology has been gaining a lot of interest in nanobiotechnology as well.…”

Section: Perspectives: Future Developments In Nanoparticle Deliverymentioning

confidence: 99%

Delivering Colloidal Nanoparticles to Mammalian Cells: A Nano–Bio Interface Perspective

Verderio

Avvakumova

Alessio

et al. 2014

Adv Healthcare Materials

View full text Add to dashboard Cite

Understanding the behavior of multifunctional colloidal nanoparticles capable of biomolecular targeting remains a fascinating challenge in materials science with dramatic implications in view of a possible clinical translation. In several circumstances, assumptions on structure-activity relationships have failed in determining the expected responses of these complex systems in a biological environment. The present Review depicts the most recent advances about colloidal nanoparticles designed for use as tools for cellular nanobiotechnology, in particular, for the preferential transport through different target compartments, including cell membrane, cytoplasm, mitochondria, and nucleus. Besides the conventional entry mechanisms based on crossing the cellular membrane, an insight into modern physical approaches to quantitatively deliver nanomaterials inside cells, such as microinjection and electro-poration, is provided. Recent hypotheses on how the nanoparticle structure and functionalization may affect the interactions at the nano-bio interface, which in turn mediate the nanoparticle internalization routes, are highlighted. In addition, some hurdles when this small interface faces the physiological environment and how this phenomenon can turn into different unexpected responses, are discussed. Finally, possible future developments oriented to synergistically tailor biological and chemical properties of nanoconjugates to improve the control over nanoparticle transport, which could open new scenarios in the field of nanomedicine, are addressed.

show abstract

“…Meanwhile, the HaloTag was optimized and a new variant was established (Ohana et al, 2009). Additional reports underline the positive rather than negative effect of this new HaloTag variant on the soluble production of recombinant enzymes (Peterson and Kwon, 2012;Sun et al, 2015).…”

Section: Introductionmentioning

confidence: 99%