Search Results (4)

ApoB-100 is a member of a large lipid transfer protein superfamily and is one of the main apolipoproteins found on low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) particles. Despite its clinical significance for the development of cardiovascular disease, there is limited information on apoB-100 structure. We have developed a novel method based on the “divide and conquer” algorithm, using PSIPRED software, by dividing apoB-100 into five subunits and 11 domains. Models of each domain were prepared using I-TASSER, DEMO, RoseTTAFold, Phyre2, and MODELLER. Subsequently, we used disuccinimidyl sulfoxide (DSSO), a new mass spectrometry cleavable cross-linker, and the known position of disulfide bonds to experimentally validate each model. We obtained 65 unique DSSO cross-links, of which 87.5% were within a 26 Å threshold in the final model. We also evaluated the positions of cysteine residues involved in the eight known disulfide bonds in apoB-100, and each pair was measured within the expected 5.6 Å constraint. Finally, multiple domains were combined by applying constraints based on detected long-range DSSO cross-links to generate five subunits, which were subsequently merged to achieve an uninterrupted architecture for apoB-100 around a lipoprotein particle. Moreover, the dynamics of apoB-100 during particle size transitions was examined by comparing VLDL and LDL computational models and using experimental cross-linking data. In addition, the proposed model of receptor ligand binding of apoB-100 provides new insights into some of its functions. Full article

A leading biotechnological advancement in the field of biocatalysis is the immobilization of enzymes on solid supports to create more stable and recyclable systems. Metal-organic frameworks (MOFs) are porous materials that have been explored as solid supports for enzyme immobilization. Composed of organic linkers and inorganic nodes, MOFs feature empty void space with large surface areas and have the ability to be modified post-synthesis. Our target enzyme system for immobilization is glucose oxidase (GOx) and chloroperoxidase (CPO). Glucose oxidase catalyzes the oxidation of glucose and is used for many applications in biosensing, biofuel cells, and food production. Chloroperoxidase is a fungal heme enzyme that catalyzes peroxide-dependent halogenation, oxidation, and hydroxylation. These two enzymes work sequentially in this enzyme system by GOx producing peroxide, which activates CPO that reacts with a suitable substrate. This study focuses on using a zirconium-based MOF, UiO-66-NH₂, to immobilize the enzyme system via crosslinking with the MOF’s amine group on the surface of the MOF. This study investigates two different crosslinkers: disuccinimidyl glutarate (DSG) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinidimide (NHS), providing stable crosslinking of the MOF to the enzymes. The two crosslinkers are used to covalently bond CPO and GOx onto UiO-66-NH₂, and a comparison of the recyclability and enzymatic activity of the single immobilization of CPO and the doubly immobilized CPO and GOx is discussed through assays and characterization analyses. The DSG-crosslinked composites displayed enhanced activity relative to the free enzyme, and all crosslinked enzyme/MOF composites demonstrated recyclability, with at least 30% of the activity being retained after four catalytic cycles. The results of this report will aid researchers in utilizing CPO as a biocatalyst that is more active and has greater recyclability. Full article

A regenerable immunoaffinity layer comprising covalently immobilized orientation-controlled antibodies was developed for use in a surface plasmon resonance (SPR) biosensor. For antibody orientation control, antibody-binding Z-domain-autodisplaying Escherichia coli (E. coli) cells and their outer membrane (OM) were utilized, and a disuccinimidyl crosslinker was employed for covalent antibody binding. To fabricate the regenerable immunoaffinity layer, capture antibodies were bound to autodisplayed Z-domains, and then treated with the crosslinker for chemical fixation to the Z-domains. Various crosslinkers, namely disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS) and poly (ethylene glycol)-ylated bis (sulfosuccinimidyl)suberate (BS(PEG)₅), were evaluated, and DSS at a concentration of 500 μM was confirmed to be optimal. The E. coli-cell-based regenerable HRP immunoassay was evaluated employing three sequential HRP treatment and regeneration steps. Then, the Oms of E. coli cells were isolated and layered on a microplate and regenerable OM-based HRP immunoassaying was evaluated. Five HRP immunoassays with four regeneration steps were found to be feasible. This regenerable, covalently immobilized, orientation-controlled OM-based immunoaffinity layer was applied to an SPR biosensor, which was capable of quantifying C-reactive protein (CRP). Five regeneration cycles were repeated using the demonstrated immunoaffinity layer with a signal difference of <10%. Full article

Numerous studies have addressed the utilization of glutaraldehyde (GA) as a homobifunctional cross-linker. However, its applicability has been impeded due to several issues, including the tendency of GA molecules to undergo polymerization. Herein, a portable urea biosensor was developed for the real-time monitoring of the flow of physiological fluids; this was achieved by using disuccinimidyl cross-linker-based urease immobilization. Urease was immobilized on a porous polytetrafluoroethylene (PTFE) solid support using different disuccinimidyl cross-linkers, namely disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS) and bis-N-succinimidyl-(pentaethylene glycol) ester (BS(PEG)₅). A urease activity test revealed that DSS exhibited the highest urease immobilizing efficiency, whereas FT-IR analysis confirmed that urease was immobilized on the PTFE membrane via DSS cross-linking. The membrane was inserted in a polydimethylsiloxane (PDMS) fluidic chamber that generated an electrochemical signal in the presence of a flowing fluid containing urea. Urea samples were allowed to flow into the urea biosensor (1.0 mL/min) and the signal was measured using chronoamperometry. The sensitivity of the DSS urea biosensor was the highest of all the trialed biosensors and was found to be superior to the more commonly used GA cross-linker. To simulate real-time monitoring in a human patient, flowing urea-spiked human serum was measured and the effective urease immobilization of the DSS urea biosensor was confirmed. The repeatability and interference of the urea biosensor were suitable for monitoring urea concentrations typically found in human patients. Full article