Steric-Deficient Oligoglycine Surrogates Facilitate Multivalent and Bifunctional Nanobody Synthesis via Combined Sortase A Transpeptidation and Click Chemistry.

Conferring multifunctional properties to proteins via enzymatic approaches has greatly facilitated recent progress in protein nanotechnology. In this regard, sortase (Srt) A transpeptidation has facilitated many of these developments due to its exceptional specificity, mild reaction conditions, and complementation with other bioorthogonal techniques, such as click chemistry. In most of these developments, Srt A is used to seamlessly tether oligoglycine-containing molecules to a protein of interest that is equipped with the enzyme's recognition sequence, LPXTG. However, the dependence on oligoglycine attacking nucleophiles and the associated cost of certain derivatives (e.g., cyclooctyne) limit the utility of this approach to lab-scale applications only. Thus, the quest to identify appropriate alternatives and understand their effectiveness remains an important area of research. This study identifies that steric and nucleophilicity-associated effects influence Srt A transpeptidation when two oligoglycine surrogates were examined. The approach was further used in complementation with click chemistry to synthesize bivalent and bifunctional nanobody conjugates for application in epithelial growth factor receptor targeting. The overall technique and tools developed here may facilitate the advancement of future nanotechnologies.

Sortase A transpeptidation produces seamless, unbranched biotinylated nanobodies for multivalent and multifunctional applications.

Exploitation of the biotin-streptavidin interaction for advanced protein engineering is used in many bio-nanotechnology applications. As such, researchers have used diverse techniques involving chemical and enzyme reactions to conjugate biotin to biomolecules of interest for subsequent docking onto streptavidin-associated molecules. Unfortunately, the biotin-streptavidin interaction is susceptible to steric hindrance and conformational malformation, leading to random orientations that ultimately impair the function of the displayed biomolecule. To minimize steric conflicts, we employ sortase A transpeptidation to produce quantitative, seamless, and unbranched nanobody-biotin conjugates for efficient display on streptavidin-associated nanoparticles. We further characterize the protein-nanoparticle complex and demonstrate its usefulness in optical microscopy and multivalent severe acute respiratory syndrome coronavirus (SARS-CoV-2) antigen interaction. The approach reported here provides a template for making novel multivalent and multifunctional protein complexes for avidity-inspired technologies.

Harnessing sortase A transpeptidation for advanced targeted therapeutics and vaccine engineering.

The engineering of potent prophylactic and therapeutic complexes has always required careful protein modification techniques with seamless capabilities. In this light, methods that favor unobstructed multivalent targeting and correct antigen presentations remain essential and very demanding. Sortase A (SrtA) transpeptidation has exhibited these attributes in various settings over the years. However, its applications for engineering avidity-inspired therapeutics and potent vaccines have yet to be significantly noticed, especially in this era where active targeting and multivalent nanomedications are in great demand. This review briefly presents the SrtA enzyme and its associated transpeptidation activity and describes interesting sortase-mediated protein engineering and chemistry approaches for achieving multivalent therapeutic and antigenic responses. The review further highlights advanced applications in targeted delivery systems, multivalent therapeutics, adoptive cellular therapy, and vaccine engineering. These innovations show the potential of sortase-mediated techniques in facilitating the development of simple plug-and-play nanomedicine technologies against recalcitrant diseases and pandemics such as cancer and viral infections.

Multivalent ACE2 engineering-A promising pathway for advanced coronavirus nanomedicine development.

The spread of coronavirus diseases has resulted in a clarion call to develop potent drugs and vaccines even as different strains appear beyond human prediction. An initial step that is integral to the viral entry into host cells results from an active-targeted interaction of the viral spike (S) proteins and the cell surface receptor, called angiotensin-converting enzyme 2 (ACE2). Thus, engineered ACE2 has been an interesting decoy inhibitor against emerging coronavirus infestation. This article discusses promising innovative ACE2 engineering pathways for current and emerging coronavirus therapeutic development. First, we provide a brief discussion of some ACE2-associated human coronaviruses and their cell invasion mechanism. Then, we describe and contrast the individual spike proteins and ACE2 receptor interactions, highlighting crucial hotspots across the ACE2-associated coronaviruses. Lastly, we address the importance of multivalency in ACE2 nanomedicine engineering and discuss novel approaches to develop and achieve multivalent therapeutic outcomes. Beyond coronaviruses, these approaches will serve as a paradigm to develop new and improved treatment technologies against pathogens that use ACE2 receptor for invasion.

Anti-SARS-CoV-1 and -2 nanobody engineering towards avidity-inspired therapeutics.

In the past two decades, the emergence of coronavirus diseases has been dire distress on both continental and global fronts and has resulted in the search for potent treatment strategies. One crucial challenge in this search is the recurrent mutations in the causative virus spike protein, which lead to viral escape issues. Among the current promising therapeutic discoveries is the use of nanobodies and nanobody-like molecules. While these nanobodies have demonstrated high-affinity interaction with the virus, the unpredictable spike mutations have warranted the need for avidity-inspired therapeutics of potent inhibitors such as nanobodies. This article discusses novel approaches for the design of anti-SARS-CoV-1 and -2 nanobodies to facilitate advanced innovations in treatment technologies. It further discusses molecular interactions and suggests multivalent protein nanotechnology and chemistry approaches to translate mere molecular affinity into avidity.

Parametric study of immobilized cellulase-polymethacrylate particle for the hydrolysis of carboxymethyl cellulose.

Biocarriers are pivotal in enhancing the reusability of biocatalyst that would otherwise be less economical for industrial application. Ever since the induction of enzymatic technology, varied materials have been assessed for their biocompatibility with enzymes of distinct functionalities. Herein, cellulase was immobilized onto polymethacrylate particles (ICP) as the biocarrier grafted with ethylenediamine (EDA) and glutaraldehyde (GA). Carboxymethyl cellulose (CMC) was used as a model substrate for activity assay. Enzyme immobilization loading was determined by quantifying the dry weight differential of ICP (pre-& post-immobilization). Cellulase was successfully demonstrated to be anchored upon ICP and validated by FTIR spectra analysis. The optimal condition for cellulase immobilization was determined to be pH 6 at 20 °C. The maximum CMCase activity was achieved at pH 5 and 50 °C. Residual activity of ∼50% was retained after three iterations and dipped to ∼18% on following cycle. Also, ICP displayed superior pH adaptability as compared to free cellulase. The specific activity of ICP was 65.14 ±â€¯1.11% relative to similar amount of free cellulase.

The workability of Escherichia coli BL21 (DE3) and Pseudomonas putida KT2440 expression platforms with autodisplayed cellulases: a comparison.

This article comparatively reports the workability of Escherichia coli BL21(DE3) and Pseudomonas putida KT2440 cell factories for the expression of three model autodisplayed cellulases (i.e., endoglucanase, BsCel5A; exoglucanase, CelK; ß-glucosidase, BglA). The differentiation of the recombinant cells was restricted to their cell growth and enzyme expression/activity attributes. Comparatively, the recombinant E. coli showed higher cell growth rates but lower enzyme activities than the recombinant P. putida. However, the endo-, exoglucanase, and ß-glucosidase on the surfaces of both cell factories showed activity over a broad range of pH (4-10) and temperature (30-100 °C). The pH and temperature optima were pH 6, 60 °C (BsCel5A); pH 6, 60-70 °C (CelK); and pH 6, 50 °C (BglA). Overall, the P. putida cell factory with autodisplayed enzymes demonstrated higher bioactivity and remarkable biochemical characteristics and thus was chosen for the saccharification of filter paper. A volumetric blend of the three cellulases with P. putida as the host yielded a ratio of 1:1:1.5 of endoglucanase, exoglucanase, and ß-glucosidase, respectively, as the optimum blend composition for filter paper degradation. At an optical density (578 nm) of 50, the blend generated a maximum sugar yield of about 0.7 mg/ml (~ 0.08 U/g) from Whatman filter paper (Ø 6 mm, ~ 2.5 mg) within 24 h.

Elucidating endotoxin-biomolecule interactions with FRET: extending the frontiers of their supramolecular complexation.

Endotoxin has been one of the topical chemical contaminants of major concern to researchers, especially in the field of bioprocessing. This major concern of researchers stems from the fact that the presence of Gram-negative bacterial endotoxin in intracellular products is unavoidable and requires complex downstream purification steps. For instance, endotoxin interacts with recombinant proteins, peptides, antibodies and aptamers and these interactions have formed the foundation for most biosensors for endotoxin detection. It has become imperative for researchers to engineer reliable means/techniques to detect, separate and remove endotoxin, without compromising the quality and quantity of the end-product. However, the underlying mechanism involved during endotoxin-biomolecule interaction is still a gray area. The use of quantitative molecular microscopy that provides high resolution of biomolecules is highly promising, hence, may lead to the development of improved endotoxin detection strategies in biomolecule preparation. Förster resonance energy transfer (FRET) spectroscopy is one of the emerging most powerful tools compatible with most super-resolution techniques for the analysis of molecular interactions. However, the scope of FRET has not been well-exploited in the analysis of endotoxin-biomolecule interaction. This article reviews endotoxin, its pathophysiological consequences and the interaction with biomolecules. Herein, we outline the common potential ways of using FRET to extend the current understanding of endotoxin-biomolecule interaction with the inference that a detailed understanding of the interaction is a prerequisite for the design of strategies for endotoxin identification and removal from protein milieus.