The Protein Journal最新文献

A phytochemical investigation of Glycyrrhiza echinata led to the isolation and structural characterization of twelve phenolic compounds. An in silico target fishing analysis identified protein tyrosine phosphatase 1B (PTP1B) as a potential biological target for these phytochemicals, prompting an in vitro evaluation of their PTP1B inhibitory activities. Gancaonin Q and licoflavone C exhibited notably low IC₅₀ values (1.61 ± 0.32 µM and 1.39 ± 0.33 µM, respectively), outperforming the reference inhibitor ursolic acid (IC₅₀ = 7.17 ± 0.69 µM), while norartocarpetin showed moderate activity (IC₅₀ = 42.41 ± 2.12 µM). Enzyme kinetic studies revealed that gancaonin Q and licoflavone C act as noncompetitive inhibitors of PTP1B. Subsequent in silico analyses supported these findings and provided mechanistic insights. Molecular docking confirmed robust binding interactions for gancaonin Q and licoflavone C at the PTP1B allosteric site. Free energy landscape (FEL) calculations indicated that both compounds stabilized the enzyme within low-energy conformations, and MM/PBSA estimations corroborated their favorable binding free energies. Molecular dynamics simulations further demonstrated the stability of the ligand-enzyme complexes, characterized by reduced structural fluctuations in comparison with the free enzyme and norartocarpetin-bound states. Finally, ADMET assessments indicated promising pharmacokinetic and toxicity profiles, with some scope for structural refinement. Overall, these results highlight gancaonin Q and licoflavone C as promising lead compounds for the development of PTP1B inhibitors with therapeutic potential.

Phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) of cestodes is considered a possible anthelmintic target because of its differential role in their hosts. In an earlier study, the recombinant PEPCK from Raillietina echinobothrida (rePEPCK) was overexpressed as inclusion bodies and was solubilized following renaturation with chemical additives, specifically L-arginine. Molecular chaperones are alternatives to chemical additives and detergents because they preserve the stability and conformation of the proteins. Hence, in this study, the recombinant rePEPCK was subcloned into the pE-SUMO vector and co-expressed along with the molecular chaperones (e.g. pG-KJE8, pG-Tf2) in Escherichia coli BL21 (DE3) cells. The protein was purified using affinity chromatography and subsequently characterized. The overexpressed rePEPCK was found to be a monomer of ~ 75 kDa. The optimum activity of the enzyme was observed in 50 mM Tris–HCl buffer at pH 7.0. In comparison, Mn²⁺ at 4.0 mM and GDP at 0.6 mM were observed to be the ideal cofactor and nucleotide, respectively. The V_max of the purified rePEPCK was found to be ~ 0.279 U/mg protein and K_m value of ~ 35.87 μM for its substrate. The turnover number (k_cat) of rePEPCK was found to be 4.7 s⁻¹ with catalytic efficiency (k_cat/K_m) 1.31 × 10⁵ M⁻¹ s⁻¹. The chaperones interacted with the key amino acids of PEPCK. This investigation explored the role of the chaperones in producing biologically active rePEPCK for its characterisation and may improve the understanding of the biochemical and biophysical properties of the enzyme as an anthelmintic target.

Resveratrol, a natural phytoalexin synthesized by certain plants in response to injury, exhibits antimicrobial properties and various medically significant effects, including anticancer and antiaging activities. Although its exact mechanism of action is still under investigation, it is believed to involve its interaction with a group of protein deacetylases known as sirtuins. Sirtuins are crucial in regulating metabolism, stress responses, and processes such as lifespan extension through caloric restriction. In this study, we report the inhibitory effect of resveratrol on the growth of Leishmania amazonensis promastigotes, highlighting its potential as a microbicide. Through fluorescence spectroscopy assays and in silico analysis, we identified and characterized the interaction between resveratrol and Sir2-related protein 1 from L. amazonensis (rLaSir2RP1). Our results demonstrate a direct interaction between resveratrol and rLaSir2RP1, characterized by a binding constant of 10⁵ M⁻¹. This interaction involves a single binding site located near a hydrophobic pocket, which includes its solely tryptophan residue.

Insulin lispro, a fast-acting insulin analogue, is widely used for the treatment of diabetes due to its rapid onset and high flexibility. This study established a three-step chromatography purification process, L fermentation broth can obtain 475 mg/L insulin lispro. Through a multi-step process involving fermentation, recovery of inclusion bodies, renaturation, enzymatic digestion, and chromatographic purification, a high-purity insulin lispro product was obtained. The final product achieved a purity of 99.7%. The high molecular weight protein was ≤ 0.1%, zinc content was 0.41%, host cell protein contamination was 2.31 ng/mg, and residual DNA was 1.57 ng/mg. Analytical results, including retention time, molecular weight, and peptide mapping, were consistent with theoretical expectations and matched the control. This purification process provides a streamlined and cost-effective method for large-scale production of insulin lispro, supporting further development of recombinant insulin analogs and offering high-quality active pharmaceutical ingredients for pharmaceutical formulations.

Plant-derived lectins are getting growing interest for their potential biomedical applications, however, there are few studies on lectins from Eucalyptus vicina fruits. The aim of the current study was to isolate and characterize a new phytolectin from Eucalyptus vicina fruits (EVL) and evaluate its immunomodulatory activity. EVL was purified through ammonium sulfate precipitation (70%) followed by ion-exchange chromatography. The purified protein appeared to have a molecular weight of around 55 kDa, determined using sodium dodecyl sulfate–polyacrylamide gel electrophoresis SDS-PAGE. EVL had strong hemagglutination activity against rabbit erythrocytes and all human blood types and displayed a high affinity against fetuin. The lectin exhibited optimal activity at alkaline pH (8–10) and was remarkably thermostable for 1 h at 70 degrees. Metal ions did not alter activity to a significant degree. The immunomodulatory effects were assessed in vivo using a carbon clearance assay, and EVL injection resulted in a dose-dependent increase in phagocytic activity. Overall, our data suggests that EVL could be a valuable candidate for research and future immunomodulatory therapies.

Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae), commonly known as the cotton mealybug, is a highly invasive pest worldwide, particularly in tropical and subtropical regions. Despite posing a significant economic threat to various agricultural crops, a promising, environmentally friendly control strategy against this pest is lacking. Furthermore, the molecular aspects of this insect pest remain understudied. This pioneering study provides the first proteome data for four different developmental stages of the cotton mealybug. A comparative analysis of differential protein expression (DEPs) among six groups revealed the highest number of DEPs (550 up-regulated and 1118 down-regulated) when comparing the egg and first nymphal stages to the second nymphal instar (fold change ≥ 2, P < 0.05). From the generated proteomics data, potential target genes were identified for cotton mealybug management. These genes were further evaluated for RNAi-based pest control and optimization of the dsRNA delivery system in cotton mealybug. Notably, RNAi-based pest management analysis revealed that dsRNA of the Ferritin-like precursor (Psfer) gene (TRINITY_DN17055_c1_g1_i1) induced significant mortality (~ 69%), followed by dsRNA of the probable cytochrome P450 6a14-like (Psp450 6a14) gene (TRINITY_DN47081_c0_g1) and odorant-binding protein 2 precursor (Psobp) gene (TRINITY_DN11547_c0_g1). This investigation proposes a potential alternative, eco-friendly strategy for managing cotton mealybug populations and related pests. Furthermore, this study provides valuable insights into the proteome of the cotton mealybug and Hemiptera, offering avenues for proteome-based identification of RNAi targets for pest management and crop improvement.

Stem cell factor (SCF) is a type of hematopoietic cytokine that functions in early hematopoietic stem cells. SCF, together with its cognate receptor c-kit, plays crucial roles in cell survival, proliferation, and differentiation. A dysregulated SCF/c-kit system is reportedly, associated strongly with various kinds of cancer in humans. However, the function and mechanism of SCF in non-small cell lung cancer (NSCLC) remains to be elucidated to date. In this context, the present study attempted to purify two different sizes of human SCF proteins and then investigate the effect of these exogenous SCFs in A549 cells. First, the homo sapiens Kit ligand (KITG) transcript variants a and b were amplified, and recombinant pET-30a vectors were constructed. Soluble His-SCF and His-SCF-2 were then expressed and purified using 0.1 mM isopropyl-β-D-1-tiogalactopiranoside (IPTG) at 25 °C and 0.3 mM IPTG at 25 °C, respectively. MTT assay was conducted next, which revealed that the purified proteins significantly promoted A549 cell activity and increased the phosphorylation of P65. Further, NF-κB pathway blockade was achieved through treatment with BAY11-7082, and it was observed to decrease the A549 cell activity promoted by the purified proteins. These findings provided insights into the biological role of SCF in the development of NSCLC.

Mammalian paraoxonase-1 (PON1) is a ~ 39.45 kDa calcium-dependent hydrolytic enzyme with potential therapeutic applications in chemical defense and cardiovascular disease. The N-terminus of PON1 is embedded in the cellular membrane, imparting to a hydrophobic character that leads to increased aggregation propensity and instability during purification. Although some advances have been made in bacterial expression hosts by using solubility-enhancing fusion tags and detergent solubilization strategies, these studies have shown that proteolytic tag removal is generally problematic. Thus, ineffective tag removal limits the bioanalytical characterization of the enzyme. Furthermore, the need for stabilizing detergents during purification limits the options for affinity-tag based methods. In this study, we demonstrate a novel affinity purification strategy by combining two solubility-enhancing fusion partners with the iCapTag™ self-removing affinity tag, where the entire purification process takes place in the presence of detergent. Optimization of purification conditions, including detergent and pH, resulted in the successful solubilization and stabilization of rePON1 at room temperature, allowing the tagless and native protein to be characterized. The results confirmed the expected catalytic efficiency and molecular weight of the enzyme. This method achieved over 95% host-cell protein impurities and more than 99.9% clearance of the host cell’s double-stranded DNA in a single-column affinity operation. This approach combines the power of affinity chromatography and facile tag removal, thereby offering a versatile and efficient alternative to produce other recombinant membrane-associated proteins, as well as additional target proteins that require challenging buffer conditions.