Expression of acid cleavable Asp-Pro linked multimeric AFP peptide in E. coli

Background Difficult to express peptides are usually produced by co-expression with fusion partners. In this case, a significant mass part of the recombinant product falls on the subsequently removed fusion partner. On the other hand, multimerization of peptides is known to improve its proteolytic stability in E. coli due to the inclusion of body formation, which is sequence specific. Thereby, the peptide itself may serve as a fusion partner and one may produce more than one mole of the desired product per mole of fusion protein. This paper proposes a method for multimeric production of a human alpha-fetoprotein fragment with optimized multimer design and processing. This fragment may further find its application in the cytotoxic drug delivery field or as an inhibitor of endogenous alpha-fetoprotein. Results Multimerization of the extended alpha-fetoprotein receptor-binding peptide improved its stability in E. coli, and pentamer was found to be the largest stable with the highest expression level. As high as 10 aspartate-proline bonds used to separate peptide repeats were easily hydrolyzed in optimized formic acid-based conditions with 100% multimer conversion. The major product was represented by unaltered functional alpha-fetoprotein fragment while most side-products were its formyl-Pro, formyl-Tyr, and formyl-Lys derivatives. Single-step semi-preparative RP-HPLC was enough to separate unaltered peptide from the hydrolysis mixture. Conclusions A recombinant peptide derived from human alpha-fetoprotein can be produced via multimerization with subsequent formic acid hydrolysis and RP-HPLC purification. The reported procedure is characterized by the lower reagent cost in comparison with enzymatic hydrolysis of peptide fusions and solid-phase synthesis. This method may be adopted for different peptide expression, especially with low amino and hydroxy side chain content.

Abstract P201: Negative Regulation Of PVN Pre-sympathetic Neuronal Activity By Resident Microglia

Hypothalamic paraventricular nucleus (PVN) is a critical integrating region in controlling peripheral sympathetic tonicity. While the vast studies have unraveled the regulatory circuits affecting PVN pre-sympathetic neurons, local factors for maintaining the homeostasis of neuronal excitability are barely understood. In the present study we investigated the role of microglia, the primary resident immune cells of the CNS, in this context. By electrophysiological recording, we found that loss of resident microglia induced an increased firing frequency and attenuated outward potassium currents in the PVN pre-sympathetic neurons, tachycardia and impaired heart rate variability. Combining the transcriptomics analysis of the PVN microglia, we identified a releasable factor, which was dominantly expressed in microglia compared to other brain parenchymal cells. ICV infusion of the recombinant peptide restored potassium currents in the PVN pre-sympathetic neurons and autonomic function in microglia-depleted mice. In summary, our results provided a novel intrinsic regulatory mechanism by which microglia suppress neuronal over excitation in physiological condition.

Comparison of Osteogenic Potentials of Dental Pulp and Bone Marrow Mesenchymal Stem Cells Using the New Cell Transplantation Platform, CellSaic, in a Rat Congenital Cleft-Jaw Model

Scaffolds stimulate cell proliferation and differentiation and play major roles in providing growth and nutrition factors in the repair of bone defects. We used the recombinant peptide Cellnest™ to prepare the three-dimensional stem cell complex, CellSaic, and evaluated whether CellSaic containing rat dental pulp stem cells (rDPSCs) was better than that containing rat bone marrow stem cells (rBMSCs). rDPSC-CellSaic or rBMSC-CellSaic, cultured with or without osteogenic induction medium, formed the experimental and control groups, respectively. Osteoblast differentiation was evaluated in vitro and transplanted into a rat model with a congenital jaw fracture. Specimens were collected and evaluated by microradiology and histological analysis. In the experimental group, the amount of calcium deposits, expression levels of bone-related genes (RUNX2, ALP, BSP, and COL1), and volume of mineralized tissue, were significantly higher than those in the control group (p < 0.05). Both differentiated and undifferentiated rDPSC-CellSaic and only the differentiated rBMSC-CellSaic could induce the formation of new bone tissue. Overall, rBMSC-CellSaic and rDPSC-CellSaic made with Cellnest™ as a scaffold, provide excellent support for promoting bone regeneration in rat mandibular congenital defects. Additionally, rDPSC-CellSaic seems a better source for craniofacial bone defect repair than rBMSC-CellSaic, suggesting the possibility of using DPSCs in bone tissue regenerative therapy.

A fructosylated peptide derived from a collagen II T cell epitope for long-term treatment of arthritis (FIA-CIA) in mice

Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease which affects primarily the joints. Peptides of several proteins have shown an effect in some experimental animal models of RA. We investigated arthritis development in male DBA/1 mice which were injected with bovine collagen II (bCII) and human fibrinogen (hFib) on days 0 and 21, leading to stable and reproducible disease induction in 100% of immunized mice (FIA-CIA). In a second study, two bCII—derived peptides were given three times in the course of 6 weeks after FIA-CIA induction to test for impact on arthritis. Mice were scored weekly for arthritis and anti-citrullinated peptide antibodies (ACPAs) were determined in the sera taken on days 0, 14, 35, 56 and 84. Histology of the hind paws was performed at the end of the experiment. Intravenous administration of peptide 90578, a novel fructosylated peptide derived from the immunodominant T cell epitope of bCII, at a dosage of 1 mg/kg resulted in significant beneficial effects on clinical outcome parameters and on the arthritis histology scores which was sustained over 12 weeks. Survival tended to be improved in peptide 90578-treated mice. Intravenous administration of pure soluble peptide 90578 without adjuvants is a promising approach to treat RA, with treatment starting at a time when ACPAs are already present. The results complement existing data on peptide “vaccination” of healthy animals, or on treatment using recombinant peptide expressing virus or complex biological compounds.

Rapid Generation of Coronaviral Immunity Using Recombinant Peptide Modified Nanodiamonds

Vaccination remains one of the most effective tools to prevent infectious diseases. To ensure that the best possible antigenic components are chosen to stimulate a cognitive immune response, boosting antigen presentation using adjuvants is common practice. Nanodiamond-based adjuvants are proposed here as a rapid and versatile platform for antigen conjugation, utilizing peptides common to different pathogenic strains and making this strategy a good candidate for a “ready-to-use” vaccine. Initiation of an inflammatory reaction with a resulting immune response is based on the ability of living organisms to entrap nanostructures such as nanodiamonds with neutrophil extracellular traps (NETs) formation. In this work, coronavirus peptide homological for MERS-CoV, fusion inhibitor, was conjugated to nanodiamonds and used to induce neutrophilic-driven self-limiting inflammation. The resulting adjuvant was safe and did not induce any tissue damage at the site of injection. Mice immunization resulted in IgG titers of ¼,000 within 28 days. Immunization of rabbits resulted in the formation of a high level of antibodies persistently present for up to 120 days after the first immunization (animal lifespan ~3 years). The peptide used for immunization proved to be reactive with sera of convalescent COVID patients, demonstrating the possibility of developing pancoronaviral vaccine candidates.

Recombinant active Peptides and their Therapeutic functions

: Recombinant active peptides are utilized as diagnostic and biotherapeutics in various maladies and as bacterial growth inhibitors in the food industry. This consequently stimulated the need for recombinant peptides' production, which resulted in about 19 approved biotech peptides of 1-100 amino acids commercially available. While most peptides have been produced by chemical synthesis, the production of lengthy and complicated peptides comprising natural amino acids has been problematic with low quantity. Recombinant peptide production has become very vital, cost-effective, simple, environmentally friendly with satisfactory yields. Several reviews have focused on discussing expression systems, advantages, disadvantages, and alternatives strategies. Additionally, the information on the antimicrobial activities and other functions of multiple recombinant peptides is challenging to access and is scattered in literature apart from the food and drug administration (FDA) approved ones. From the reports that come to our knowledge, there is no existing review that offers substantial information on recombinant active peptides developed by researchers and their functions. This review provides an overview of some successfully produced recombinant active peptides of ≤100 amino acids by focusing on their antibacterial, antifungal, antiviral, anticancer, antioxidant, antimalarial, and immune-modulatory functions. It also elucidates their modes of expression that could be adopted and applied in future investigations. We expect that the knowledge available in this review would help researchers involved in recombinant active peptide development for therapeutic uses and other applications.

Unique role and vulnerability of EP300 KIX domain in small-cell lung cancer

EP300 (E1A binding protein p300) is a versatile transcription co-activator important in cell proliferation and differentiation. The gene EP300 is frequently mutated in diverse cancer types, including small-cell lung cancer (SCLC). While it is widely believed that these mutations result in loss of EP300 function, the impact on SCLC pathogenesis remains largely unknown. Here we demonstrate that mutant EP300 variants lacking histone acetyltransferase (HAT) domain accelerate tumor development in autochthonous mouse models of SCLC. However, unexpectedly, complete knockout of Ep300 suppresses tumor development and inhibits proliferation of both human and mouse SCLC cells. Genetic dissection of EP300 domains identifies kinase-inducible domain (KID)-interacting (KIX) domain, specifically its interaction with transcription factors such as CREB1 and MYB, as the determinant of pro-tumorigenic activity. Blockade of the KIX-mediated protein interactions using a small molecule and a recombinant peptide mimicking the KIX-binding sequences of EP300-interacting partners inhibits the growth of SCLC cells. These findings identify domain-specific roles of EP300 in SCLC and unique vulnerability of the EP300 KIX domain to potential therapeutics.

Effectiveness of a New Recombinant antiGnRH Vaccine for Immunocastration in Bulls

Castration by surgical techniques is common in livestock; however, post-surgery complications and concerns for animal wellbeing have created a need for new non-invasive alternatives. The objective of this study was to evaluate immunocastration in bulls using antigen GnRX G/Q; a recombinant peptide proved to be effective in laboratory and companion animals. A nine-month trial with 80 9-month-old Normand x Hereford bulls, kept in a pastured system, was conducted. The herd was divided in half with 40 bulls surgically castrated (SC) and 40 castrated by immunization against GnRH (IC). The antigen was injected on days 0 and 40 of the experiment. After the second dose, the IC group had elevated GnRH antibodies and decreased testosterone levels (below 5 ng/mL) that were maintained for 23 weeks. At slaughter on day 190, the immunocastrated group obtained a higher weight, hot carcass, and dressing percentage than the SC group. There was no difference in pH, color of meat, fat coverage, cooking loss, or tenderness between groups. The bulls showed no inflammatory reaction at the injection site or adverse side effects from the vaccine. Our results demonstrate that immunocastration with GnRX G/Q is an efficient and safe alternative to surgical castration in livestock. Additional work evaluating antigen effects over a longer period is needed to validate commercial viability.