Protein-based conjugates are valuable constructs for a variety of applications. Conjugation of proteins to fluorophores is commonly used to study their cellular localization and the protein–protein interactions. Modification of therapeutic proteins with either polymers or cytotoxic moieties greatly enhances their pharmacokinetics or potency. To label a protein of interest, conventional direct chemical reaction with the side-chains of native amino acids often yields heterogeneously modified products. This renders their characterization complicated, requires difficult separation steps and may impact protein function. Although modification can also be achieved via the insertion of unnatural amino acids bearing bioorthogonal functional groups, these methods can have lower protein expression yields, limiting large scale production. As a site-specific modification method, enzymatic protein labelling is highly efficient and robust under mild reaction conditions. Significant progress has been made over the last five years in modifying proteins using enzymatic methods for numerous applications, including the creation of clinically relevant conjugates with polymers, cytotoxins or imaging agents, fluorescent or affinity probes to study complex protein interaction networks, and protein-linked materials for biosensing. This review summarizes developments in enzymatic protein labelling over the last five years for a panel of ten enzymes, including sortase A, subtiligase, microbial transglutaminase, farnesyltransferase, N-myristoyltransferase, phosphopantetheinyl transferases, tubulin tyrosin ligase, lipoic acid ligase, biotin ligase and formylglycine generating enzyme.

/pdf/recent-progress-in-enzymatic-protein-labelling-techniques-3bchcly0vc.pdf

Recent progress in enzymatic protein labelling techniques and their applications

Within the materials science community, proteins with cage-like architectures are being developed as versatile nanoscale platforms for use in protein nanotechnology. Much effort has been focused on the functionalization of protein cages with biological and non-biological moieties to bring about new properties of not only individual protein cages, but collective bulk-scale assemblies of protein cages. In this review, we report on the current understanding of protein cage assembly, both of the cages themselves from individual subunits, and the assembly of the individual protein cages into higher order structures. We start by discussing the key properties of natural protein cages (for example: size, shape and structure) followed by a review of some of the mechanisms of protein cage assembly and the factors that influence it. We then explore the current approaches for functionalizing protein cages, on the interior or exterior surfaces of the capsids. Lastly, we explore the emerging area of higher order assemblies created from individual protein cages and their potential for new and exciting collective properties.

/pdf/protein-cage-assembly-across-multiple-length-scales-dpc8z1tt12.pdf

Protein cage assembly across multiple length scales.

As mimicking natural virus structures, virus-like particles (VLPs) have evolved to become a widely accepted technology used for humans which are safe, highly efficacious, and profitable. Several remarkable advantages have been achieved to revolutionize the molecule delivery for diverse applications in nanotechnology, biotechnology, and medicine. Here, the rational structure design, manufacturing process, functionalization strategy, and emerging applications of VLPs is reviewed. The situation and challenges in the VLP engineering, the key development orientation, and future applications have been discussed. To develop a good VLP design concept, the virus/VLP-host interactions need to be examined and the screening methods of the VLP stabilization factors need to be established. The functionalization toolbox can be expanded to fabricate smart, robust, and multifunctional VLPs. Novel robust VLP manufacturing platforms are required to deliver vaccines in resource-poor regions with a significant reduction in the production time and cost. The future applications of VLPs are always driven by the development of emerging technologies and new requirements of modern life.

Virus-Like Particle Engineering: From Rational Design to Versatile Applications.

The hepatitis B virus-like particle (HBV VLP) is an attractive protein nanoparticle platform due to the availability of 240 modification sites for engineering purposes. Although direct protein insertion into the surface loop has been demonstrated, this decoration strategy is restricted by the size of the inserted protein moieties. Meanwhile, larger proteins can be decorated using chemical conjugations; yet these approaches perturb the integrity of more delicate proteins and can unfavorably orient the proteins, impairing active surface display. Herein, we aim to create a robust and highly modular method to produce smart HBV-based nanodevices by using the SpyCatcher/SpyTag system, which allows a wide range of peptides and proteins to be conjugated directly and simply onto the modified HBV capsids in a controlled and biocompatible manner. Our technology allows the modular surface modification of HBV VLPs with multiple components, which provides signal amplification, increased targeting avidity, and high therapeutic payload incorporation. We have achieved a yield of over 200 mg/L for these engineered HBV VLPs and demonstrated the flexibility of this platform in both biosensing and drug delivery applications. The ability to decorate over 200 nanoluciferases per VLP improved detection signal by over 1500-fold, such that low nanomolar levels of thrombin could be detected by the naked eye. Meanwhile, a dimeric prodrug-activating enzyme was loaded without cross-linking particles by coexpressing orthogonally labeled monomers. This along with a epidermal growth factor receptor-binding peptide enabled tunable uptake of HBV VLPs into inflammatory breast cancer cells, leading to efficient suicide enzyme delivery and cell killing.

Modular Hepatitis B Virus-like Particle Platform for Biosensing and Drug Delivery.

Sortases are enzymes occurring in the cell wall of Gram-positive bacteria. Sortase A (SrtA), the best studied sortase class, plays a key role in anchoring surface proteins with the recognition sequence LPXTG covalently to oligoglycine units of the bacterial cell wall. This unique transpeptidase activity renders SrtA attractive for various purposes and motivated researchers to study multiple in vivo and in vitro ligations in the last decades. This ligation technique is known as sortase-mediated ligation (SML) or sortagging and developed to a frequently used method in basic research. The advantages are manifold: extremely high substrate specificity, simple access to substrates and enzyme, robust nature and easy handling of sortase A. In addition to the ligation of two proteins or peptides, early studies already included at least one artificial (peptide equipped) substrate into sortagging reactions – which demonstrates the versatility and broad applicability of SML. Thus, SML is not only a biology-related technique, but has found prominence as a major interdisciplinary research tool. In this review, we provide an overview about the use of sortase A in interdisciplinary research, mainly for protein modification, synthesis of protein–polymer conjugates and immobilization of proteins on surfaces.

/pdf/broadening-the-scope-of-sortagging-5elgdn1n56.pdf

Broadening the scope of sortagging

Sortase-Mediated Ligation as a Modular Approach for the Covalent Attachment of Proteins to the Exterior of the Bacteriophage P22 Virus-like Particle

Engineering Virus Like Particles Towards Directing Immunologic Responses

The electrical conductivity, temperature coefficient of resistance (TCR), and electrical low frequency noise in VOx thin films were investigated. The electrical conduction is found to be dominated by Variable Range Hopping (VRH). Phenomenological relations between resistivity, TCR, and low frequency noise were determined for VOx films over a wide range of resistivities. It was observed that both TCR and noise increase monotonically with resistivity, as expected for VRH conduction.

Conductivity and low frequency noise in VOx thin films

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Sortase-Mediated Ligation as a Modular Approach for the Covalent Attachment of Proteins to the Exterior of the Bacteriophage P22 Virus-like Particle

Engineering Virus Like Particles Towards Directing Immunologic Responses

Conductivity and low frequency noise in VOx thin films