Comparison between different conditions for the incorporation of foreign proteins into Autographa californica multiple polyhedrovirus polyhedra for biotechnological purposes.

The occlusion bodies of Autographa californica multiple nucleopolyhedrovirus are proteinaceous formations with significant biotechnological potential owing to their capacity to integrate foreign proteins through fusion with polyhedrin, their primary component. However, the strategy for successful heterologous protein inclusion still requires further refinement. In this study, we conducted a comparative assessment of various conditions to achieve the embedding of recombinant proteins within polyhedra. Two baculoviruses were constructed: AcPHGFP (polh+), with GFP as a fusion to wild type (wt) polyhedrin and AcΔPHGFP (polh+), with GFP fused to a fragment corresponding to amino acids 19 to 110 of polyhedrin. These baculoviruses were evaluated by infecting Sf9 cells and stably transformed Sf9, Sf9POLH, and Sf9POLHE44G cells. The stably transformed cells contributed another copy of wt or a mutant polyhedrin, respectively. Polyhedra of each type were isolated and characterized by classical methods. The fusion PHGFP showed more-efficient incorporation into polyhedra than ΔPHGFP in the three cell lines assayed. However, ΔPHGFP polyhedron yields were higher than those of PHGFP in Sf9 and Sf9POLH cells. Based on an integral analysis of the studied parameters, it can be concluded that, except for the AcΔPHGFP/Sf9POLHE44G combination, deficiencies in one factor can be offset by improved performance by another. The combinations AcPHGFP/Sf9POLHE44G and AcΔPHGFP/Sf9POLH stand out due to their high level of incorporation and the large number of recombinant polyhedra produced, respectively. Consequently, the choice between these approaches becomes dependent on the intended application.

Development of hybrid biomicroparticles: cellulose exposing functionalized fusion proteins.

BACKGROUND: One of the leading current trends in technology is the miniaturization of devices to the microscale and nanoscale. The highly advanced approaches are based on biological systems, subjected to bioengineering using chemical, enzymatic and recombinant methods. Here we have utilised the biological affinity towards cellulose of the cellulose binding domain (CBD) fused with recombinant proteins. RESULTS: Here we focused on fusions with 'artificial', concatemeric proteins with preprogrammed functions, constructed using DNA FACE™ technology. Such CBD fusions can be efficiently attached to micro-/nanocellulose to form functional, hybrid bionanoparticles. Microcellulose (MCC) particles were generated by a novel approach to enzymatic hydrolysis using Aspergillus sp. cellulase. The interaction between the constructs components - MCC, CBD and fused concatemeric proteins - was evaluated. Obtaining of hybrid biomicroparticles of a natural cellulose biocarrier with proteins with therapeutic properties, fused with CBD, was confirmed. Further, biological tests on the hybrid bioMCC particles confirmed the lack of their cytotoxicity on 46BR.1 N fibroblasts and human adipose derived stem cells (ASCs). The XTT analysis showed a slight inhibition of the proliferation of 46BR.1 N fibroblasts and ACSs cells stimulated with the hybrid biomicroparticles. However, in both cases no changes in the morphology of the examined cells after incubation with the hybrid biomicroparticles' MCC were detected. CONCLUSIONS: Microcellulose display with recombinant proteins involves utilizing cellulose, a natural polymer found in plants, as a platform for presenting or displaying proteins. This approach harnesses the structural properties of cellulose to express or exhibit various recombinant proteins on its surface. It offers a novel method for protein expression, presentation, or immobilization, enabling various applications in biotechnology, biomedicine, and other fields. Microcellulose shows promise in biomedical fields for wound healing materials, drug delivery systems, tissue engineering scaffolds, and as a component in bio-sensors due to its biocompatibility and structural properties.

Oxygen imaging of hypoxic pockets in the mouse cerebral cortex.

Consciousness is lost within seconds upon cessation of cerebral blood flow. The brain cannot store oxygen, and interruption of oxidative phosphorylation is fatal within minutes. Yet only rudimentary knowledge exists regarding cortical partial oxygen tension (Po2) dynamics under physiological conditions. Here we introduce Green enhanced Nano-lantern (GeNL), a genetically encoded bioluminescent oxygen indicator for Po2 imaging. In awake behaving mice, we uncover the existence of spontaneous, spatially defined "hypoxic pockets" and demonstrate their linkage to the abrogation of local capillary flow. Exercise reduced the burden of hypoxic pockets by 52% compared with rest. The study provides insight into cortical oxygen dynamics in awake behaving animals and concurrently establishes a tool to delineate the importance of oxygen tension in physiological processes and neurological diseases.

Engineered Chimera Protein Constructs to Facilitate the Production of Heterologous Transmembrane Proteins in E. coli.

To delve into the structure-function relationship of transmembrane proteins (TMPs), robust protocols are needed to produce them in a pure, stable, and functional state. Among all hosts that express heterologous TMPs, E. coli has the lowest cost and fastest turnover. However, many of the TMPs expressed in E. coli are misfolded. Several strategies have been developed to either direct the foreign TMPs to E. coli's membrane or retain them in a cytosolic soluble form to overcome this deficiency. Here, we summarize protein engineering methods to produce chimera constructs of the desired TMPs fused to either a signal peptide or precursor maltose binding protein (pMBP) to direct the entire construct to the periplasm, therefore depositing the fused TMP in the plasma membrane. We further describe strategies to produce TMPs in soluble form by utilizing N-terminally fused MBP without a signal peptide. Depending on its N- or C-terminus location, a fusion to apolipoprotein AI can either direct the TMP to the membrane or shield the hydrophobic regions of the TMP, maintaining the soluble form. Strategies to produce G-protein-coupled receptors, TMPs of Mycobacterium tuberculosis, HIV-1 Vpu, and other TMPs are discussed. This knowledge could increase the scope of TMPs' expression in E. coli.

SARS-CoV-2 spike-FLIPr fusion protein plus lipidated FLIPr protects against various SARS-CoV-2 variants in hamsters.

Vaccine-induced mucosal immunity and broad protective capacity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remain inadequate. Formyl peptide receptor-like 1 inhibitory protein (FLIPr), produced by Staphylococcus aureus, can bind to various Fcγ receptor subclasses. Recombinant lipidated FLIPr (rLF) was previously found to be an effective adjuvant. In this study, we developed a vaccine candidate, the recombinant Delta SARS-CoV-2 spike (rDS)-FLIPr fusion protein (rDS-F), which employs the property of FLIPr binding to various Fcγ receptors. Our study shows that rDS-F plus rLF promotes rDS capture by dendritic cells. Intranasal vaccination of mice with rDS-F plus rLF increases persistent systemic and mucosal antibody responses and CD4/CD8 T-cell responses. Importantly, antibodies induced by rDS-F plus rLF vaccination neutralize Delta, Wuhan, Alpha, Beta, and Omicron strains. Additionally, rDS-F plus rLF provides protective effects against various SARS-CoV-2 variants in hamsters by reducing inflammation and viral loads in the lung. Therefore, rDS-F plus rLF is a potential vaccine candidate to induce broad protective responses against various SARS-CoV-2 variants.IMPORTANCEMucosal immunity is vital for combating pathogens, especially in the context of respiratory diseases like COVID-19. Despite this, most approved vaccines are administered via injection, providing systemic but limited mucosal protection. Developing vaccines that stimulate both mucosal and systemic immunity to address future coronavirus mutations is a growing trend. However, eliciting strong mucosal immune responses without adjuvants remains a challenge. In our study, we have demonstrated that using a recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-formyl peptide receptor-like 1 inhibitory protein (FLIPr) fusion protein as an antigen, in combination with recombinant lipidated FLIPr as an effective adjuvant, induced simultaneous systemic and mucosal immune responses through intranasal immunization in mice and hamster models. This approach offered protection against various SARS-CoV-2 strains, making it a promising vaccine candidate for broad protection. This finding is pivotal for future broad-spectrum vaccine development.

Targeted Delivery of HSP70 to Tumor Cells via Supramolecular Complex Based on HER2-Specific DARPin9_29 and the Barnase:Barstar Pair.

(1) Background: We have previously shown that the use of an artificial supramolecular two-component system based on chimeric recombinant proteins 4D5scFv-barnase and barstar-heat shock protein 70 KDa (HSP70) allows targeted delivery of HSP70 to the surface of tumor cells bearing HER2/neu antigen. In this work, we studied the possibility to using DARPin9_29-barnase as the first targeting module recognizing HER2/neu-antigen in the HSP70 delivery system. (2) Methods: The effect of the developed systems for HSP70 delivery to human carcinomas SK-BR-3 and BT474 cells hyperexpressing HER2/neu on the activation of cytotoxic effectors of the immune cells was studied in vitro. (3) Results: The results obtained by confocal microscopy and cytofluorimetric analysis confirmed the binding of HSP70 or its fragment HSP70-16 on the surface of the treated cells. In response to the delivery of HSP70 to tumor cells, we observed an increase in the cytolytic activity of different cytotoxic effector immune cells from human peripheral blood. (4) Conclusions: Targeted modification of the tumor cell surface with molecular structures recognized by cytotoxic effectors of the immune system is among new promising approaches to antitumor immunotherapy.

Ribosomal frameshifting at normal codon repeats recodes functional chimeric proteins in human.

Ribosomal frameshifting refers to the process that ribosomes slip into +1 or -1 reading frame, thus produce chimeric trans-frame proteins. In viruses and bacteria, programmed ribosomal frameshifting can produce essential trans-frame proteins for viral replication or regulation of other biological processes. In humans, however, functional trans-frame protein derived from ribosomal frameshifting is scarcely documented. Combining multiple assays, we show that short codon repeats could act as cis-acting elements that stimulate ribosomal frameshifting in humans, abbreviated as CRFS hereafter. Using proteomic analyses, we identified many putative CRFS events from 32 normal human tissues supported by trans-frame peptides positioned at codon repeats. Finally, we show a CRFS-derived trans-frame protein (HDAC1-FS) functions by antagonizing the activities of HDAC1, thus affecting cell migration and apoptosis. These data suggest a novel type of translational recoding associated with codon repeats, which may expand the coding capacity of mRNA and diversify the regulation in human.

Expression and purification of epinecidin-1 variant (Ac-Var-1) by acid cleavage.

The demand for massive quantities of therapeutic active antimicrobial peptides (AMPs) is high due to their potential as alternatives to antibiotics. However, each antimicrobial peptide has unique properties, necessitating distinct synthesis and purification strategies for their large-scale production. In this study, we bio-synthesized and purified a functional enhanced variant of the AMP epinecidin-1, known as Ac-Var-1 (acid-cleavable variant-1). To generate the active peptide, we cloned the gene for Ac-Var-1 with acid-cleavable site (aspartic acid-proline) into the pET-32a expression vector, purified the fusion protein by His tag enrichment chromatography, and performed acid cleavage to release the active Ac-Var-1 peptide. After acid cleavage, the active Ac-Var-1 was purified and characterized by SDS-PAGE and mass spectrometry. The results from both techniques provided confirmation of the intactness of the purified Ac-Var-1. The Ac-Var-1 inhibited the growth of pathogenic Escherichia coli and Staphylococcus aureus. KEY POINTS : • Epinecidin-1 is a well-known antimicrobial peptide having multipotential bioactivities. • Epinecidin-1 variant is developed via the site-directed mutagenesis method to improve its structural stability and bioactivity. • AC-Var-1 development is an economical and easy method to remove peptide from tag protein.

Designing Ubiquitin-like protease 1 (Ulp1) based nano biocatalysts: A promising technology for SUMO fusion proteins.

The SUMO proteases (Ulps), a group of cysteine proteases, are well known for their efficient ability to perform structure-based cleavage of SUMO tag from the protein of interest and generation of biotherapeutics with authentic N-terminus. However, the stability of Ulps has remained a challenge for the economical production of difficult-to-produce proteins in E. coli. Therefore, the present study aimed to establish the methodology for developing stable S. pombe Ulp1 preparation using different enzyme immobilization strategies. The whole-cell biocatalyst developed using the Pir1 anchor protein of Pichia cleaved the SUMO tag within 24 h of reaction incubation. The chemical immobilization using commercial epoxy and amino methacrylate beads significantly enhanced the operational reusability of SpUlp1 up to 24 cycles. Silica beads further improved the repetitive usage of the immobilized enzyme for 65 cycles. The SpUlp1 immobilization on laboratory-developed chitosan-coated iron oxide nanoparticles exhibited more than 90 % cleavage of SUMO tag from different substrates even after 100 consecutive reactions. Moreover, an effective SUMO tag removal was observed within 10 min of incubation. The operational stability of the immobilized enzyme was confirmed in a pH range of 5 to 13. The spherical nature of nanoparticles was confirmed by FESEM and TEM results. The successful chitosan coating and subsequent activation with glutaraldehyde were established via FT-IR. Furthermore, HRTEM, SAED, and XRD proved the crystalline nature of nanoparticles, while VSM confirmed the superparamagnetic behavior.

Synthesis of biologically active proteins as L6KD-SUMO fusions forming inclusion bodies in Escherichia coli.

A new platform has been developed to facilitate the production of biologically active proteins and peptides in Escherichia coli. The platform includes an N-terminal self-associating L6 KD peptide fused to the SUMO protein (small ubiquitin-like protein modifier) from the yeast Saccharomyces cerevisiae, which is known for its chaperone activity. The target proteins are fused at the C termini of the L6 KD-SUMO fusions, and the resulting three-component fusion proteins are synthesized and self-assembled in E. coli into so-called active inclusion bodies (AIBs). In vivo, the L6 KD-SUMO platform facilitates the correct folding of the target proteins and directs them into AIBs, greatly simplifying their purification. In vitro, the platform facilitates the effective separation of AIBs by centrifugation and subsequent target protein release using SUMO-specific protease. The properties of the AIBs were determined using five proteins with different sizes, folding efficiencies, quaternary structure, and disulfide modifications. Electron microscopy shows that AIBs are synthesized in the form of complex fibrillar structures resembling "loofah sponges" with unusually thick filaments. The obtained results indicate that the new platform has promising features and could be developed to facilitate the synthesis and purification of target proteins and protein complexes without the use of renaturation.

Theranostic role of 89Zr- and 177Lu-labeled aflibercept in breast cancer.

PURPOSE: Triple-negative breast cancer (TNBC) has a poor prognosis due to the absence of effective therapeutic targets. Vascular endothelial growth factor (VEGF) family are expressed in 30-60% of TNBC, therefore providing potential therapeutic targets for TNBC. Aflibercept (Abe), a humanized recombinant fusion protein specifically bound to VEGF-A, B and placental growth factor (PIGF), has proven to be effective in the treatment in some cancers. Therefore, 89Zr/177Lu-labeled Abe was investigated for its theranostic role in TNBC. METHODS: Abe was radiolabeled with 89Zr and 177Lu via the conjugation of chelators. Flow cytometry and cell immunofluorescent staining were performed to evaluate the binding affinity of Abe. Sequential PET imaging and fluorescent imaging were conducted in TNBC tumor bearing mice following the injection of 89Zr-labeled Abe and Cy5.5-labeled Abe. Treatment study was performed after the administration of 177Lu-labeled Abe. Tumor volume and survival were monitored and SPECT imaging and biodistribution studies were conducted. Safety evaluation was performed including body weight, blood cell measurement, and hematoxylin-eosin (H&E) staining of major organs. Expression of VEGF and CD31 was tested by immunohistochemical staining. Dosimetry was estimated using the OLINDA software. RESULTS: FITC-labeled Abe showed a strong binding affinity to VEGF in TNBC 4T1 cells and HUVECs by flow cytometry and cell immunofluorescence. Tumor uptake of 89Zr-labeled Abe peaked at 120 h (SUVmax = 3.2 ± 0.64) and persisted before 168 h (SUVmax = 2.54 ± 0.42). The fluorescence intensity of the Cy5.5-labeled Abe group surpassed that of the Cy5.5-labeled IgG group, implying that Cy5.5-labeled Abe is a viable candidate monitoring in vivo tumor targeting and localization. 177Lu-labeled Abe (11.1 MBq) served well as the therapeutic component to suppress tumor growth with standardized tumor volume at 16 days, significantly smaller than PBS group (about 815.66 ± 3.58% vs 3646.52 ± 11.10%, n = 5, P < 0.01). Moreover, SPECT images confirmed high contrast between tumors and normal organs, indicating selective tumor uptake of 177Lu-labeled Abe. No discernible abnormalities in blood cells, and no evident histopathological abnormality observed in liver, spleen, and kidney. Immunohistochemical staining showed that 177Lu-labeled Abe effectively inhibited the expression of VEGF and CD31 of tumor, suggesting that angiogenesis may be suppressed by 177Lu-labeled Abe. The whole-body effective dose for an adult human was estimated to be 0.16 mSv/MBq. CONCLUSION: 89Zr/177Lu-labeled Abe could be a TNBC-specific marker with diagnostic value and provide insights into targeted therapy in the treatment of TNBC. Further clinical evaluation and translation may be of high significance for TNBC.

Novel Anti-Mesothelin Nanobodies and Recombinant Immunotoxins with Pseudomonas Exotoxin Catalytic Domain for Cancer Therapeutics.

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated Pseudomonas exotoxin (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in Escherichia coli, achieving high solubility and purity post-purification. In vitro cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC50 values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.

Use of the selected metal-dependent enzymes for exploring applicability of human annexin A1 as a purification tag.

Several fusion tags have been developed for non-chromatographic fusion protein purification. Previously, we identified that human annexin A1 as a novel N-terminal purification tag was used for purifying the fusion proteins produced in Escherichia coli through precipitation in 10 mM Ca2+ buffer, and redissolution of the precipitate in 15 mM EDTA buffer. In this work, we selected four metal-dependent enzymes including E. coli 5-aminolevulinate dehydratase, yeast 3-hydroxyanthranilate 3,4-dioxygenase, maize serine racemase and copper amine oxidase for investigating the annexin A1 tag applicability. Fusion of the His6-tag or the enzyme changed the behavior of precipitation-redissolution. The relatively high recovery yields of three tagged enzymes with the improved purities were obtained through two rounds of purification, whereas low recovery yield of the annexin A1 tagged maize amine oxidase was prepared. The added EDTA displayed different abilities to redissolve the fusion proteins precipitates in two precipitation-redissolution cycles. It inactivated three enzymes and obviously inhibited the activity of the fused maize serine racemase. Based on current findings, we believe that four enzymes could be applied for evaluating applicability of the proteins or peptides as affinity tags for chromatographic purification in a calcium dependent manner.

Construction of a Collagen-like Protein Based on Elastin-like Polypeptide Fusion and Evaluation of Its Performance in Promoting Wound Healing.

In the healing of wounds, human-like collagen (hCol) is essential. However, collagen-based composite dressings have poor stability in vivo, which severely limits their current therapeutic potential. Based on the above, we have developed a recombinant fusion protein named hCol-ELP, which consists of hCol and an elastin-like peptide (ELP). Then, we examined the physicochemical and biological properties of hCol-ELP. The results indicated that the stability of the hCol-ELP fusion protein exhibited a more compact and homogeneous lamellar microstructure along with collagen properties, it was found to be significantly superior to the stability of free hCol. The compound hCol-ELP demonstrated a remarkable capacity to induce the proliferation and migration of mouse embryo fibroblast cells (NIH/3T3), as well as enhance collagen synthesis in human skin fibroblasts (HSF) when tested in vitro. In vivo, hCol-ELP demonstrated significant enhancements in healing rate and a reduction in the time required for scab removal, thereby exhibiting a scar-free healing effect. The findings provide a crucial theoretical foundation for the implementation of an hCol-ELP protein dressing in fields associated with the healing of traumatic injuries.

sTREM2 is a plasma biomarker for human NASH and promotes hepatocyte lipid accumulation.

BACKGROUND: Pathogenetic mechanisms of the progression of NAFL to advanced NASH coupled with potential noninvasive biomarkers and novel therapeutic targets are active areas of investigation. The recent finding that increased plasma levels of a protein shed by myeloid cells -soluble Triggering Receptor Expressed on Myeloid cells 2 (sTREM2) -may be a biomarker for NASH has received much interest. We aimed to test sTREM2 as a biomarker for human NASH and investigate the role of sTREM2 in the pathogenesis of NASH. METHODS: We conducted studies in both humans (comparing patients with NASH vs. NAFL) and in mice (comparing different mouse models of NASH) involving measurements of TREM2 gene and protein expression levels in the liver as well as circulating sTREM2 levels in plasma. We investigated the pathogenetic role of sTREM2 in hepatic steatosis using primary hepatocytes and bone marrow derived macrophages. RESULTS: RNA sequencing analysis of livers from patients with NASH or NAFL as well as livers from 2 mouse models of NASH revealed elevated TREM2 expression in patients/mice with NASH as compared with NAFL. Plasma levels of sTREM2 were significantly higher in a well-characterized cohort of patients with biopsy-proven NASH versus NAFL (area under receiver-operating curve 0.807). Mechanistic studies revealed that cocultures of primary hepatocytes and macrophages with an impaired ability to shed sTREM2 resulted in reduced hepatocyte lipid droplet formation on palmitate stimulation, an effect that was counteracted by the addition of exogenous sTREM2 chimeric protein. Conversely, exogenous sTREM2 chimeric protein increased lipid droplet formation, triglyceride content, and expression of the lipid transporter CD36 in hepatocytes. Furthermore, inhibition of CD36 markedly attenuated sTREM2-induced lipid droplet formation in mouse primary hepatocytes. CONCLUSIONS: Elevated levels of sTREM2 due to TREM2 shedding may directly contribute to the pathogenesis of NAFLD by promoting hepatocyte lipid accumulation, as well as serving as a biomarker for distinguishing patients with NASH versus NAFL. Further investigation of sTREM2 as a clinically useful diagnostic biomarker and of the therapeutic effects of targeting sTREM2 in NASH is warranted.

Evolutionary Engineering a Larger Porin Using a Loop-to-Hairpin Mechanism.

In protein evolution, diversification is generally driven by genetic duplication. The hallmarks of this mechanism are visible in the repeating topology of various proteins. In outer membrane ß-barrels, duplication is visible with ß-hairpins as the repeating unit of the barrel. In contrast to the overall use of duplication in diversification, a computational study hypothesized evolutionary mechanisms other than hairpin duplications leading to increases in the number of strands in outer membrane ß-barrels. Specifically, the topology of some 16- and 18-stranded ß-barrels appear to have evolved through a loop to ß-hairpin transition. Here we test this novel evolutionary mechanism by creating a chimeric protein from an 18-stranded ß-barrel and an evolutionarily related 16-stranded ß-barrel. The chimeric combination of the two was created by replacing loop L3 of the 16-stranded barrel with the sequentially matched transmembrane ß-hairpin region of the 18-stranded barrel. We find the resulting chimeric protein is stable and has characteristics of increased strand number. This study provides the first experimental evidence supporting the evolution through a loop to ß-hairpin transition.

Production of recombinant human long-acting IL-18 binding protein: inhibitory effect on ulcerative colitis in mice.

Interleukin-18 binding protein (IL-18BP) is a natural IL-18 inhibitor in vivo, which can effectively neutralize IL-18 and inhibit the inflammatory signaling pathway induced by IL-18, thus playing an anti-inflammatory role. Traditional production methods primarily rely on eukaryotic animal cell expression systems, which often entail complex processes, lower yields, and increase production costs. In this study, we present a novel approach for expressing IL-18BP fusion protein using the Escherichia coli (E. coli) system. The N-terminal segment of IL-18BP was fused with the small ubiquitin-related modifier (SUMO) tag, enabling soluble expression, while the C-terminal segment was fused with the human IgG1 Fc fragment to prolong its in vivo lifespan. Through screening, we obtained a high-expression engineering strain from a single colony and developed optimized protocols for fermentation and purification of the recombinant SUMO-IL-18BP-Fc protein. The SUMO tag was subsequently cleaved using SUMO protease, and the purified recombinant human IL-18BP-Fc (rhIL-18BP-Fc) exhibited a purity exceeding 90% with a yield of 1 g per liter of bacterial solution. The biological activities and underlying mechanisms of rhIL-18BP-Fc were evaluated using cell lines and a mouse model. Our results demonstrated that rhIL-18BP-Fc effectively inhibited IL-18-stimulated IFN-γ production in KG-1a cells in vitro and ameliorated DSS-induced ulcerative colitis in mice. In conclusion, we successfully employed the SUMO fusion system to achieve high-level production, soluble expression, and prolonged activity of rhIL-18BP-Fc in E. coli. These findings lay the groundwork for future large-scale industrial production and pharmaceutical development of rhIL-18BP-Fc protein. KEY POINTS: • Effective expression, fermentation, and purification of bioactive rhIL-18BP-Fc protein in E. coli. • The rhIL-18BP-Fc protein has a great potential for the therapy of ulcerative colitis by inhibiting the expression of inflammatory factors.

Biodistribution of recombinant factor IX, extended half-life recombinant factor IX Fc fusion protein, and glycoPEGylated recombinant factor IX in hemophilia B mice.

Extended half-life recombinant FIX (rFIX) molecules have been generated to reduce the dosing burden and increase the protection of patients with hemophilia B. Clinical pharmacology studies with recombinant factor IX Fc fusion protein (rFIXFc) report a similar initial peak plasma recovery to that of rFIX, but with a larger volume of distribution. Although the pegylation of N9-GP results in a larger plasma recovery, there is a smaller volume of distribution, suggesting less extravasation of the latter drug. In this study, we set out to compare the biodistribution and tissue localization of rFIX, rFIXFc, and glycoPEGylated rFIX in a hemophilia B mouse model. Radiolabeled rFIX, rFIXFc, and rFIX-GP were employed in in vivo single-photon emission computed tomography imaging (SPECT/CT), microautoradiography (MARG), and histology to assess the distribution of FIX reagents over time. Immediately following injection, vascularized tissues demonstrated intense signal irrespective of FIX reagent. rFIX and rFIXFc were retained in joint and muscle areas through 5 half-lives, unlike rFIX-GP (assessed by SPECT). MARG and immunohistochemistry showed FIX agents localized at blood vessels among tissues, including liver, spleen, and kidney. Microautoradiographs, as well as fluorescent-labeled images of knee joint areas, demonstrated retention over time of FIX signal at the trabecular area of bone. Data indicate that rFIXFc is similar to rFIX in that it distributes outside the plasma compartment and is retained in certain tissues over time, while also retained at higher plasma levels. Overall, data suggest that Fc fusion does not impede the extravascular distribution of FIX.

Molecular reshaping of phage-displayed Interleukin-2 at beta chain receptor interface to obtain potent super-agonists with improved developability profiles.

Interleukin-2 (IL-2) engineered versions, with biased immunological functions, have emerged from yeast display and rational design. Here we reshaped the human IL-2 interface with the IL-2 receptor beta chain through the screening of phage-displayed libraries. Multiple beta super-binders were obtained, having increased receptor binding ability and improved developability profiles. Selected variants exhibit an accumulation of negatively charged residues at the interface, which provides a better electrostatic complementarity to the beta chain, and faster association kinetics. These findings point to mechanistic differences with the already reported superkines, characterized by a conformational switch due to the rearrangement of the hydrophobic core. The molecular bases of the favourable developability profile were tracked to a single residue: L92. Recombinant Fc-fusion proteins including our variants are superior to those based on H9 superkine in terms of expression levels in mammalian cells, aggregation resistance, stability, in vivo enhancement of immune effector responses, and anti-tumour effect.

Robust production of active Ulp1 (SUMO protease) from inclusion bodies.

High yield purification of Ulp1 is required during the isolation and purification of SUMO-tagged recombinant proteins. However, when expressed as a soluble protein, Ulp1 is toxic to E. coli host cells and most of the protein forms inclusion bodies. The extraction of insoluble Ulp1 followed by its purification and refolding into its active form is a lengthy and costly procedure. In our present study, we developed a simple, cost effective procedure for the large scale production of active Ulp1 that can be used for industrial scale requirements.