Assembly of Protein Building Blocks Using a Short Synthetic Peptide

Combining proteins or their defined domains offers new enhanced functions. Conventionally, two proteins are either fused into a single polypeptide chain by recombinant means or chemically cross-linked. However, these strategies can have drawbacks such as poor expression (recombinant fusions) or aggregation and inactivation (chemical cross-linking), especially in the case of large multifunctional proteins. We developed a new linking method which allows site-oriented, noncovalent, yet irreversible stapling of modified proteins at neutral pH and ambient temperature. This method is based on two distinct polypeptide linkers which self-assemble in the presence of a specific peptide staple allowing on-demand and irreversible combination of protein domains. Here we show that linkers can either be expressed or be chemically conjugated to proteins of interest, depending on the source of the proteins. We also show that the peptide staple can be shortened to 24 amino acids still permitting an irreversible combination of functional proteins. The versatility of this modular technique is demonstrated by stapling a variety of proteins either in solution or to surfaces

Accreditation of Biosafe Clinical-Grade Human Embryonic Stem Cells According to Chinese Regulations

Human embryonic stem cells (hESCs) are promising in regenerative medicine. Although several hESC-based clinical trials are under way, a widely accepted standard of clinical-grade cells remains obscure. To attain a completely xeno-free clinical-grade cell line, the system must be free of xenogenic components, the cells must have a comprehensive set of functions, and good manufacturing practice conditions must be used. In this study, following these criteria, we successfully derived two hESC lines, which were thereby considered clinical-grade embryonic stem cells . In addition to the primary capacity for pluripotency, these two cell lines were efficiently differentiated into various types of clinical-grade progeny. Importantly, the cells were recognized by the National Institutes for Food and Drug Control of China for further eligible accreditation. These data indicate that we have established completely xeno-free clinical-grade hESC lines and their derivatives, which will be valuable for the foundation of an international standard for clinical-grade cells for therapy

Cloning and Expression of a Novel Target Fusion Protein and its Application in Anti-Tumor Therapy

Backgrounds: Epidermal growth factor (EGF) is a 53 amino acid polypeptide and its receptor EGFR is an established therapeutic target for anti-tumor therapy. Two major categories of EGFR-targeted drugs include monoclonal antibodies (mAbs) and small molecular tyrosine kinase inhibitors (TKIs). However, drug resistance occurs in a significant proportion of patients due to EGFR mutations. Since EGFR can maintain activation while abrogating the activity of mAbs or TKIs, or bypass signaling functions while successfully circumventing the EGF-EGFR switch, developing new mechanism-based inhibitors is necessary. Methods: In this study, based on the principle of tumor immunotherapy, a recombinant protein pLLO-hEGF was constructed. The N-terminal portion contains three immunodominant epitopes from listeriolysin O (LLO) and the C-terminal includes EGF. To use EGF as a target vector to recognize EGFR-expressing cancer cells, immunodominant epitopes could enhance immunogenicity of tumor cells for immune cell activation and attack. Results: Recombinant protein pLLO-hEGF was successfully expressed and showed strong affinity to cancer cells. Also, pLLO-hEGF could significantly stimulate human lymphocyte proliferation and the lymphocytes demonstrated enhanced killing potency in EGFR-expressing cancer cells in vitro and in vivo. Conclusion: This study can provide novel strategies and directions in tumor biotherapy

A novel approach of jet polishing for interior surface of small-grooved components using three developed setups

It is a challenge to polish the interior surface of an additively manufactured component with complex structures and groove sizes less than 1 mm. Traditional polishing methods are disabled to polish the component, meanwhile keeping the structure intact. To overcome this challenge, small-grooved components made of aluminum alloy with sizes less than 1 mm were fabricated by a custom-made printer. A novel approach to multi-phase jet (MPJ) polishing is proposed, utilizing a self-developed polisher that incorporates solid, liquid, and gas phases. In contrast, abrasive air jet (AAJ) polishing is recommended, employing a customized polisher that combines solid and gas phases. After jet polishing, surface roughness ( Sa ) on the interior surface of grooves decreases from pristine 8.596 μ m to 0.701 μ m and 0.336 μ m via AAJ polishing and MPJ polishing, respectively, and Sa reduces 92% and 96%, correspondingly. Furthermore, a formula defining the relationship between linear energy density and unit defect volume has been developed. The optimized parameters in additive manufacturing are that linear energy density varies from 0.135 J mm ^−1 to 0.22 J mm ^−1 . The unit area defect volume achieved via the optimized parameters decreases to 1/12 of that achieved via non-optimized ones. Computational fluid dynamics simulation results reveal that material is removed by shear stress, and the alumina abrasives experience multiple collisions with the defects on the heat pipe groove, resulting in uniform material removal. This is in good agreement with the experimental results. The novel proposed setups, approach, and findings provide new insights into manufacturing complex-structured components, polishing the small-grooved structure, and keeping it unbroken

Stapling of the botulinum type A protease to growth factors and neuropeptides allows selective targeting of neuroendocrine cells

Precise cellular targeting of macromolecular cargos has important biotechnological and medical implications. Using a recently established ‘protein stapling’ method, we linked the proteolytic domain of botulinum neurotoxin type A (BoNT/A) to a selection of ligands to target neuroendocrine tumor cells. The botulinum proteolytic domain was chosen because of its well-known potency to block the release of neurotransmitters and hormones. Among nine tested stapled ligands, the epidermal growth factor was able to deliver the botulinum enzyme into pheochromocytoma PC12 and insulinoma Min6 cells; ciliary neurotrophic factor was effective on neuroblastoma SH-SY5Y and Neuro2A cells, whereas corticotropin-releasing hormone was active on pituitary AtT-20 cells and the two neuroblastoma cell lines. In neuronal cultures, the epidermal growth factor- and ciliary neurotrophic factor-directed botulinum enzyme targeted distinct subsets of neurons whereas the whole native neurotoxin targeted the cortical neurons indiscriminately. At nanomolar concentrations, the retargeted botulinum molecules were able to inhibit stimulated release of hormones from tested cell lines suggesting their application for treatments of neuroendocrine disorders

Efficient generation of mouse ESCs-like pig induced pluripotent stem cells

Dear Editor, Porcine induced pluripotency stem cells (piPSCs) are promised in basic research, animal husbandry and regenerative medicine. However, the efficiency of the piPSCs induction has been low and the generated piPSCs varied in cell morphology and cell characteristics. Here we report a novel approach to improve efficiency of piPSCs generation. The induced piPSCs are dome-shaped mouse embryonic stem cells (ESCs)-like and display molecular properties of mouse ESCs. Electroporation study reveals that mouse ESCs-like status facilitates genetic manipulating of piPSCs. Importantly, we demonstrate that the domed piPSC colonies are more suitable as donor cells for nuclear transfer (NT) to generate reconstructed embryos than those flattened piPSCs. The potential applications of the newly generated piPSCs in ungulate pluripotent research are discussed