The silkworm-baculovirus expression vector system (silkworm-BEVS), using Bombyx mori nucleopolyhedrovirus (BmNPV) and silkworm larvae or pupae, has been used as a cost-effective expression system for the production of various recombinant proteins. Recently, several gene knockouts in baculoviruses have been shown to improve the productivity of recombinant proteins. However, the gene editing of the baculovirus genome (approximately 130kb) remains challenging and time-consuming. In this study, we sought to further enhance the productivity of the silkworm-BEVS by synthesizing and gene editing the BmNPV bacmid from plasmids containing fragments of BmNPV genomic DNA using a two-step Golden Gate Assembly (GGA). The BmNPV genome, divided into 19 fragments, was amplified by PCR and cloned into the plasmids. From these initial plasmids, four intermediate plasmids containing the BmNPV genomic DNA were constructed by GGA with the type IIS restriction enzyme BsaI. Subsequently, the full-length bacmid was successfully synthesized from the four intermediate plasmids by GGA with another type IIS restriction enzyme PaqCI with a high efficiency of 97.2%. Furthermore, this methodology enabled the rapid and straightforward generation of the BmNPV bacmid lacking six genes, resulting in the suppression of degradation of recombinant proteins expressed in silkworm pupae. These results indicate that the BmNPV bacmid can be quickly and efficiently edited using only simple cloning techniques and enzymatic reactions, marking a significant advancement in the improvement of the silkworm-BEVS.

At present, biopharmaceuticals have received extensive attention from the society, among which recombinant proteins have a good growth trend and a large market share. Chinese hamster ovary (CHO) cells are the preferred mammalian system to produce glycosylated recombinant protein drugs. A highly efficient and stable cell screening method needs to be developed to obtain more and useful recombinant proteins. Limited dilution method, cell sorting, and semi-solid medium screening are currently the commonly used cell cloning methods. These methods are time-consuming and labor-intensive, and they have the disadvantage of low clone survival rate. Here, a method based on semi-solid medium was developed to screen out high-yielding and stable cell line within 3 weeks to improve the screening efficiency. The semi-solid medium was combined with an expression vector containing red fluorescent protein (RFP) for early cell line development. In accordance with the fluorescence intensity of RFP, the expression of upstream target gene could be indicated, and the fluorescence intensity was in direct proportion to the expression of upstream target gene. In conclusion, semi-solid medium combined with bicistronic expression vector provides an efficient method for screening stable and highly expressed cell lines.

Current medical countermeasures (MCMs) for nerve agent poisoning have limited efficacy, and can cause serious adverse effects, prompting the requirement for new broad-spectrum therapeutics. Human plasma-derived butyrylcholinseterase (huBChE) is a promising novel bioscavenger MCM which has shown potential in animal studies, however, is economically prohibitive to manufacture at scale. This study addresses current challenges for the economical production of a bioactive and long-acting recombinant huBChE (rBChE) in mammalian cells by being the first to directly compare novel rBChE design strategies. These include co-expression of a proline rich attachment domain (PRAD) and fusion of BChE with a protein partner. Additionally, a pre-purification screening method developed in this study enables parallel comparison of the expression efficiency, activity and broad-spectrum binding to nerve agents for ten novel rBChE molecular designs. All designed rBChE demonstrated functionality to act as broad-spectrum MCMs to G, V and A series nerve agents. Expression using the ExpiCHO™ Max protocol provided greatest expression levels and activity for all constructs, with most rBChE expressing poorly in Expi293™. Fc- or hSA-fused rBChE significantly outperformed constructs designed to mimic huBChE, including PRAD-BChE, and proved an effective strategy to significantly improve enzyme activity and expression. Choice of protein partner, directionality and the addition of a linker also impacted fusion rBChE activity and expression. Overall, hSA fused rBChE provided greatest expression yield and activity, with BChE-hSA the best performing construct. The purified and characterized BChE-hSA demonstrated similar functionality to huBChE to be inhibited by GD, VX and A-234, supporting the findings of the pre-screening study and validating its capacity to assess and streamline the selection process for rBChE constructs in a cost-effective manner. Collectively, these outcomes contribute to risk mitigation in early-stage development, providing a systematic method to compare rBChE designs and a focus for future development.

Natural biomaterials, particularly fibrous proteins, are extensively utilized in skin tissue engineering. However, their application is impeded by batch-to-batch variance, limited chemical or physical versatility, and environmental concerns. Recent advancements in gene editing and fermentation technology have catalyzed the emergence of recombinant fibrous protein biomaterials, which are gaining traction in skin tissue engineering. The modular and highly customizable nature of recombinant synthesis enables precise control over biomaterial design, facilitating the incorporation of multiple functional motifs. Additionally, recombinant synthesis allows for a transition from animal-derived sources to microbial sources, thereby reducing endotoxin content and rendering recombinant fibrous protein biomaterials more amenable to scalable production and clinical use. In this review, we provide an overview of prevalent recombinant fibrous protein biomaterials (collagens, elastin, silk proteins and their chimeric derivatives) used in skin tissue engineering (STE) and compare them with their animal-derived counterparts. Furthermore, we discuss their applications in STE, along with the associated challenges and future prospects.

Maternal vaccinations administered prior to conception or during pregnancy enhance the immune protection of newborn infants against many pathogens. A feasibility experiment was conducted to determine if monkeys can be used to model the placental transfer of maternal antibody against SARS-CoV-2. Six adult rhesus monkeys were immunized with adjuvanted recombinant-protein antigens comprised of receptor-binding domain human IgG1-Fc fusion proteins (RBD-Fc) containing protein sequences from the ancestral-Wuhan or Gamma variants. The female monkeys mounted robust and sustained anti-SARS-CoV-2 antibody responses. Blood samples collected from their infants after delivery verified prenatal transfer of high levels of spike-specific IgG, which were positively correlated with maternal IgG titers at term. In addition, an in vitro test of ACE2 neutralization indicated that the infants\' IgG demonstrated antigen specificity, reflecting prior maternal immunization with either Wuhan or Gamma-variant antigens. All sera showed stronger ACE2-RBD binding inhibition when variants in the assay more closely resembled the vaccine RBD sequence than with more distantly related variants (i.e., Delta and Omicron). Monkeys are a valuable animal model for evaluating new vaccines that can promote maternal and infant health. Further, the findings highlight the enduring nature and safety of the immune protection elicited by an adjuvanted recombinant RBD-Fc vaccine.

The L-gulonolactone oxidase enzyme (GULO) catalyzes the last step of L-ascorbic acid (vitamin C) biosynthesis. This enzymatic activity is lost in primates. The full-length rat GULO has been previously produced in plants and demonstrated to be active. In this study, we compared the activity of two variants of GULO produced in Escheriachia coli cells, full-length rat GULO (fGULO) and its C-terminal catalytic domain (cGULO). The expression and purification of the recombinant proteins were optimized, and their biological activity was confirmed by two methods, the GULO activity assay in the protein extracts and the \'in-gel\' staining for GULO activity. Both variants of recombinant GULO were biologically active in both assays. However, cGULO is more promising than fGULO for ascorbic acid production because it is more efficiently produced by bacteria. Furthermore, the optimal activities of the fGULO and cGULO recombinant proteins were observed at pH 7 and 6.5, and at temperatures of 40 and 30 °C, respectively. Kinetic studies revealed that at low substrate concentrations, Km values for fGULO and cGULO were 53.5 ± 5 and 42 ± 6.3 µM, respectively.

OBJECTIVE: The anticancer properties of recombinant α-luffin (LUF) are wellestablished. However, the cytotoxic effects of encapsulating LUF within niosomes on the SKBR3 breast cancer cell line have yet to be explored. Our study aimed to investigate whether this encapsulation strategy could improve cytotoxic effects.
METHODS: Alpha-luffin was expressed, purified, and refolded. Then, this protein was utilized to craft an optimal formulation, guided by experimental design. In this work, we have explored various physicochemical properties, including particle size, polydispersity index, zeta potential, morphology, entrapment efficiency, drug release and kinetics, storage stability, and FTIR spectroscopy. Additionally, we have assessed the cellular uptake and cytotoxic effect of the optimized niosome formulation on the SKBR3 breast cancer cell line.
RESULTS: The optimized niosome exhibited a mean diameter of 315±6.4 nm (DLS). Successful encapsulation of LUF into regularly shaped, spherical niosomes was achieved, with an encapsulation efficiency of 73.45±2.4%. Notably, Niosomal LUF (NLUF) exhibited significantly increased cytotoxicity against SKBR3 cells.
CONCLUSIONS: These findings suggest that niosomes loaded with LUF hold promise as a potential treatment strategy for breast cancer.

Controllable regulatory elements, like inducible, titratable promoters, are highly desired in synthetic biology toolboxes. A set of previously developed erythritol-inducible promoters along with an engineered Yarrowia lipolytica host strain were shown to be a very potent expression platform. In this study, we push the previously encountered limits of the synthetic promoters\' titratability (by the number of upstream motifs) by using a compatible transcription factor, Euf1, as the promoter titrator. Overexpression of spliced EUF1 turned out to be very efficient in promoting expression from the compatible promoter, however, the erythritol-inducible character of the promoter was then lost. Analysis of the EUF1\'s splicing pattern suggests that the intron removal is promoted in the presence of erythritol, but is not dependent on it. The 3D structures of spliced vs unspliced Euf1 were modeled, and ligand binding strength was calculated and compared. Furthermore, the EUF1-dependent expression profile under different chemical stimulants was investigated. Depletion of carbon source was identified as the significant factor upregulating the expression from the Euf1-dependent promoter (2- to 10-fold). Considering these findings and transcriptomics data, a new mechanism of the Euf1-regulated promoter action is proposed, involving a \"catabolite repression\" transcription factor-Adr1, both acting on the same ERY-inducible promoter.

BACKGROUND: Widespread resistance of insect pests to insecticides and transgenic crops in the field is a significant challenge for sustainable agriculture, and calls for the development of novel alternative strategies to control insect pests. One potential resource for the discovery of novel insecticidal molecules is natural toxins, particularly those derived from the venoms of insect predators.
RESULTS: In this study, we identified three insecticidal proteinaceous toxins from the venom glands (VGs) of the predatory stink bug, Arma custos (Hemiptera: Asopinae). Transcriptomic analysis of A. custos VGs revealed 151 potentially secreted VG-rich venom proteins. Three VG-rich venom proteins (designated AcVP1 ~ 3) were produced by overexpression in Escherichia coli. Injection of the recombinant proteins into tobacco cutworm (Spodoptera litura) larvae showed that all of the three recombinant proteins caused paralysis, liquefaction and death. Injection of recombinant proteins into rice brown planthopper (Nilaparvata lugens) nymphs showed higher insecticidal activities, among which a trypsin (AcVP2) caused 100% mortality postinjection at 1.27 pmol mg-1 body weight.
CONCLUSIONS: A natural toolkit for the discovery of insecticidal toxins from predatory insects has been revealed by the present study. © 2024 Society of Chemical Industry.

Entamoeba nuttalli is genetically the closest to Entamoeba histolytica, the causative agent of human amebiasis. E. nuttalli is found in Macaca species, exhibiting no symptoms while potentially virulent. Using comparative genomics of Entamoeba species, we identified a gene encoding an E. nuttalli-specific protein containing 42 repeats of an octapeptide (PTORS). In the present study, we analyzed the genes in E. nuttalli strains derived from various geographic locations and host species. Sequence analysis of genomic DNA from four strains indicated 43, 44, and 48 repeat types in addition to 42 repeats and remarkable genetic diversity in the repeat region, although all nucleotide substitutions were synonymous. In contrast, the sequences of the N-terminal side region and C-terminus were identical among the strains. Monoclonal antibodies prepared against recombinant PTORS were reactive to the repeat regions but not to the N-terminal side regions. Polyclonal antibodies did not react with the N-terminal region, demonstrating that the repeat region had higher antigenicity. Analysis using synthetic peptides revealed that the two repeats of the octapeptide functioned as epitopes. Immunofluorescence microscopy using monoclonal antibodies demonstrated the surface localization of PTORS. These results suggest that the repeat region of PTORS plays an important role in host-parasite interactions.