Genetically engineering encapsulin protein cage nanoparticle as a SCC-7 cell targeting optical nanoprobe

Background - Protein cage nanoparticles are promising nanoplatform candidates for efficient delivery systems of diagnostics and/or therapeutics because of their uniform size and structure as well as high biocompatibility and biodegradability. Encapsulin protein cage nanoparticle is used to develop a cell-specific targeting optical nanoprobe. Results - FcBPs are genetically inserted and successfully displayed on the surface of encapsulin to form FcBP-encapsulin. Selectively binding of FcBP-encapsulin to SCC-7 is visualized with fluorescent microscopy. Conclusions - Encapsulin protein cage nanoparticle is robust enough to maintain their structure at high temperature and easily acquires multifunctions on demand through the combination of genetic and chemical modifications.ope

Surface plasmon resonance: a versatile technique for biosensor applications

Surface plasmon resonance (SPR) is a label-free detection method which has emerged during the last two decades as a suitable and reliable platform in clinical analysis for biomolecular interactions. The technique makes it possible to measure interactions in real-time with high sensitivity and without the need of labels. This review article discusses a wide range of applications in optical-based sensors using either surface plasmon resonance (SPR) or surface plasmon resonance imaging (SPRI). Here we summarize the principles, provide examples, and illustrate the utility of SPR and SPRI through example applications from the biomedical, proteomics, genomics and bioengineering fields. In addition, SPR signal amplification strategies and surface functionalization are covered in the review.open1

Directing ricin-based immunotoxins with targeting affibodies and KDEL signal peptide to cancer cells effectively induces apoptosis and tumor suppression

<jats:title>Abstract</jats:title><jats:p>The plant toxin ricin, especially its cytotoxic A chain (RTA), can be genetically engineered with targeting ligands to develop specific anti-cancer recombinant immunotoxins (RITs). Here, we used affibody molecules targeting two cancer biomarkers, the receptors HER2 and EGFR, along with the KDEL signal peptide to construct two cancer-specific ricin-based RITs, HER2Afb-RTA-KDEL and EGFRAfb-RTA-KDEL. The affibodies successfully provided target-specificity and subsequent receptor-mediated endocytosis and the KDEL signal peptide routed the RITs through the retrograde transport pathway, effectively delivering RTA to the cytosol as well as avoiding the alternate recycling pathway that typical cancer cells frequently have. The in vivo efficacy of RITs was enhanced by introducing the albumin binding domain (AlBD) to construct AlBD/HER2Afb/RTA-KDEL. Systemic administration of AlBD-containing RITs to tumor-bearing mice significantly suppressed tumor growth without any noticeable side-effects. Collectively, combining target-selective affibody molecules, a cytotoxic RTA, and an intracellularly designating peptide, we successfully developed cancer-specific and efficacious ricin-based RITs. This approach can be applied to develop novel protein-based ???magic bullets??? to effectively suppress tumors that are resistant to conventional anti-cancer drugs.</jats:p> <jats:p><jats:bold>Graphical Abstract</jats:bold></jats:p&gt

Fabrication of uniform layer-by-layer assemblies with complementary protein cage nanobuilding blocks via simple His-tag/metal recognition

A capsid-forming enzyme, lumazine synthase isolated from hyperthermophile Aquifex aeolicus (AaLS), is prepared and utilized as a template for constructing nanobuilding blocks to fabricate uniform layer-by-layer (LbL) assemblies. Two functionally complementary AaLS protein cage nanoparticles (PCNs) are generated either by genetically introducing His-tags on the surface of wild-type AaLS PCNs or by chemically attaching metal chelates (Ni-NTA moiety) to the surface of cysteine-bearing AaLS PCNs individually. The multivalent displays of His-tags (AaLS-His6 PCN) and Ni-NTA ligands (AaLS-NTA-Ni PCN) on the surface of each complementary AaLS PCN are successfully demonstrated by mass spectrometric and surface plasmon resonance analyses. By using these two complementary AaLS PCNs, uniform LbL assemblies are constructed via simple recognition between His-tags and metal chelates without the aid of additional binding mediators. This approach illustrates the potential of fabricating uniform nanostructures using protein-based hybrid functional nanobuilding blocks.close3

Lactate oxidase/catalase-displaying nanoparticles efficiently consume lactate in the tumor microenvironment to effectively suppress tumor growth

<jats:title>Abstract</jats:title><jats:p>The aggressive proliferation of tumor cells often requires increased glucose uptake and excessive anaerobic glycolysis, leading to the massive production and secretion of lactate to form a unique tumor microenvironment (TME). Therefore, regulating appropriate lactate levels in the TME would be a promising approach to control tumor cell proliferation and immune suppression. To effectively consume lactate in the TME, lactate oxidase (LOX) and catalase (CAT) were displayed onto <jats:italic>Aquifex aeolicus</jats:italic> lumazine synthase protein nanoparticles (AaLS) to form either AaLS/LOX or AaLS/LOX/CAT. These complexes successfully consumed lactate produced by CT26 murine colon carcinoma cells under both normoxic and hypoxic conditions. Specifically, AaLS/LOX generated a large amount of H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> with complete lactate consumption to induce drastic necrotic cell death regardless of culture condition. However, AaLS/LOX/CAT generated residual H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub>, leading to necrotic cell death only under hypoxic condition similar to the TME. While the local administration of AaLS/LOX to the tumor site resulted in mice death, that of AaLS/LOX/CAT significantly suppressed tumor growth without any severe side effects. AaLS/LOX/CAT effectively consumed lactate to produce adequate amounts of H<jats:sub>2</jats:sub>O<jats:sub>2</jats:sub> which sufficiently suppress tumor growth and adequately modulate the TME, transforming environments that are favorable to tumor suppressive neutrophils but adverse to tumor-supportive tumor-associated macrophages. Collectively, these findings showed that the modular functionalization of protein nanoparticles with multiple metabolic enzymes may offer the opportunity to develop new enzyme complex-based therapeutic tools that can modulate the TME by controlling cancer metabolism.</jats:p> <jats:p><jats:bold>Graphical Abstract</jats:bold></jats:p&gt

A variant of green fluorescent protein exclusively deposited to active intracellular inclusion bodies

Background: Inclusion bodies (IBs) were generally considered to be inactive protein deposits and did not hold any attractive values in biotechnological applications. Recently, some IBs of recombinant proteins were confirmed to show their functional properties such as enzyme activities, fluorescence, etc. Such biologically active IBs are not commonly formed, but they have great potentials in the fields of biocatalysis, material science and nanotechnology. Results: In this study, we characterized the IBs of DL4, a deletion variant of green fluorescent protein which forms active intracellular aggregates. The DL4 proteins expressed in Escherichia coli were exclusively deposited to IBs, and the IBs were estimated to be mostly composed of active proteins. The spectral properties and quantum yield of the DL4 variant in the active IBs were almost same with those of its native protein. Refolding and stability studies revealed that the deletion mutation in DL4 didn't affect the folding efficiency of the protein, but destabilized its structure. Analyses specific for amyloid-like structures informed that the inner architecture of DL4 IBs might be amorphous rather than well-organized. The diameter of fluorescent DL4 IBs could be decreased up to 100-200 nm by reducing the expression time of the protein in vivo. Conclusions: To our knowledge, DL4 is the first GFP variant that folds correctly but aggregates exclusively in vivo without any self-aggregating/assembling tags. The fluorescent DL4 IBs have potentials to be used as fluorescent biomaterials. This study also suggests that biologically active IBs can be achieved through engineering a target protein itself.open0