The protein journal最新文献

Glycerol kinase (GK) is a key part of glycerol metabolism. It connects the metabolic pathways for lipids and carbohydrates by phosphorylating glycerol to glycerol-3-phosphate in an ATP-dependent reaction. This is essential for maintaining carbohydrate homeostasis, plasma glycerol withdrawal, and the utilization of glycerol by different tissues. Together, these processes impact glucose uptake and lipid metabolism. This review discusses the structure of GK, highlights the implications of mutations in the primary sequence, and provides insights on the roles of the various functional domains in the GK-catalyzed reaction. It also discussed the roles of GK in glycerol metabolism, energy production, and its connections with various cellular pathways and disease conditions. The proper regulation of GK activity is crucial, reflecting its critical role in various important cellular processes. Therefore, its regulation has been analyzed from the gene level to posttranslational modification and has implications for GK-linked disease. Separately, the critical role of this enzyme in some disease-causing organisms made it a promising target for inhibitor development. We here explore the current state of GK inhibitor research and discuss strategies for their development. Challenges in GK inhibitor research are identified, and approaches such as high-throughput screening, structure-based drug design, and computational modelling for discovering novel inhibitors are reviewed. Finally, the review highlights critical areas for further research, including the role of GK in synthetic biology and tumour development, among others.

SARS-CoV-2 consists of the spike (S) protein which plays an important role in mediating the entry of virus into the host and it mainly consists of two subunits which are functionally different from each other. The first subunit S1, is involved in binding to the host receptor such as ACE2, and the second subunit S2, is involved in facilitating the viral membrane fusion with that of host membrane. Despite extensive research on the S1 domain due to its immunodominance and variability, the structurally conserved S2 domain remains relatively understudied. Recognizing the potential of S2 in modulating host responses, this study focuses on its recombinant expression, purification, and functional impact. The S2 coding region was cloned into the pGEX2TK plasmid to produce protein, which is a GST fusion-based protein and thereafter, the expression was done in BL21(DE3) strain of Escherichia coli cells. To enhance yield as well as solubility, protein expression was induced at a reduced temperature of 16 °C, minimizing aggregation and degradation. The fusion protein was purified via glutathione affinity chromatography, yielding high-purity S2 suitable for downstream applications. The S2 protein upon transfection into HEK293 and WI-38 mammalian cells leads to the expression of downregulated insulin-like growth factor 1 receptor (IGF-1R), as measured with the help of protein analysis. In order to highlight the role in pathogenesis of COVID-19 through modulation of cellular receptor and for the intervention of therapeutics, S2 can likely be considered a potential target.

D-p-hydroxyphenyl glycine (D-PHPG) is a D-amino acid used as an intermediate in the synthesis of semi-synthetic antibiotics. It is synthesized from hydantoin derivatives through two sequential enzymatic reactions involving D-hydantoinase (D-hase) and D-carbamoylase (D-case). Although whole-cell biocatalysis of D-PHPG is cost-effective, its efficiency suffers from transport obstacles, intracellular degradation, and limited substrate solubility. This study utilized a bacterial surface display system to express D-hase and D-case in Escherichia coli for D-PHPG production. Enzyme production optimization was carried out in two stages. Initially, key factors influencing cell density during co-culture were identified through culture media and fermentation parameters screening using the Plackett-Burman design, followed by optimization with the D-optimal method. Next, induction parameters were fine-tuned using response surface methodology. The optimal culture medium was found to contain glycerol (12 g/L) and yeast extract (15 g/L) under optimal induction conditions (0.17 mM IPTG, OD₆₀₀ of 1.3, and 21 °C). These conditions achieved an OD₆₀₀ of 15.6, with expression levels of 20.18% for D-case and 20.82% for D-hase. Scaling up in a stirred tank bioreactor resulted in an OD₆₀₀ of 32.15, with D-hase and D-case expression levels increasing to 25.8 and 24.2%, respectively, and enzymatic activities improving by 2.83 times for D-case and 3.42 times for D-hase. The optimized co-culture approach under optimized induction conditions achieved a conversion yield of 95% and a D-PHPG production yield of 90%. The study results showed that the suggested fermentation conditions will contribute to future scale-up studies aimed at improving enzyme activities for other surface protein production.

An essential modulator of cell growth and division is the evolutionarily conserved kinase (S6K1). It is triggered by certain stimulants, including amino acids, insulin, and other growth hormones. The Akt/phosphatidylinositol 3-kinase pathway's downstream effector, the serine/threonine kinase S6K1, is consistently activated in a variety of cancer types. Rho family guanosine triphosphate (GTPase) activation and actin filament cross-linking are two of S6K1's roles. The p70 (∆2-146/∆CT104) S6K is a truncated variant of p70S6 kinase, created by removing 146 amino acids from the N-terminal and 104 amino acids from the C-terminal end of the original protein, resulting in a total of 275 amino acids. The p70 (∆2-146/∆CT104) S6K was effectively expressed in the E. coli BL21 (DE3)pLysS strain after being cloned in E. coli DH5α. A rabbit polyclonal anti-GST antibody had been employed during Western blot analysis throughout the protein's production and purification process. Protein purification was achieved by affinity chromatography using glutathione resin-agarose beads, and chromatography onto a spin concentration column was performed. Rabbit polyclonal anti-(p70S6Kinase and GST) antibodies confirmed the presence of the purified protein.

Over the past three years, AlphaFold-a deep learning-based protein structure prediction system-has transformed structural biology by providing near-experimental accuracy models directly from amino acid sequences. This narrative review synthesizes applications reported in the 2022-2025 literature across human, microbial, and viral systems, drawing on peer-reviewed studies as our data source. Representative examples include modeling of SARS-CoV-2 spike and nucleocapsid proteins in virology, assisting cryo-EM interpretation of bacterial ribosomal and membrane-protein complexes in microbiology, and refining conformational hypotheses for human GPCRs in biomedicine. Across these cases, AlphaFold predictions have complemented experimental workflows by accelerating hypothesis generation, improving model fitting within ambiguous density regions (poorly resolved areas of cryo-EM maps), and guiding mutagenesis strategies to probe dynamic conformational states. We also summarize recent method extensions: AlphaFold-Multimer improves multi-chain complex assembly prediction, while molecular dynamics (MD) simulations augment AlphaFold's static models by sampling conformational flexibility and testing stability. Despite these advances, important limitations remain-particularly for intrinsically disordered regions, protein-ligand and protein-cofactor interactions, and very large or transient assemblies-and current community benchmarks indicate that approximately one-third of residues may lack atomistic precision, underscoring uncertainty in flexible or modified segments. Framed within a clear chronological window and evidence base, our analysis highlights both the practical impact and the remaining challenges of integrating AlphaFold with experiment, outlining priorities where further methodological innovation and orthogonal validation are needed.

Although biological drugs have been considered as one of the effective and growing therapeutic approaches in the pharmaceutical industry in recent decades, the largest concern about them is the insufficient stability and rapid degradation in the bloodstream due to their structural nature. One of the effective methods for increasing the circulating half-life of peptide and protein drugs is the addition of half-life-extending tags, which prevent both the degradation of the biological drug and its glomerular filtration by various mechanisms, thereby increasing its half-life. This review focuses on peptide and protein tags used to enhance the pharmacokinetic profiles of biological drugs by increasing their half-life. It discusses various tags, including HSA (Human Serum Albumin), ABD (Albumin Binding Domain), DARPINS (Designed Ankyrin Repeat proteins), XTEN, CTP (Carboxy Terminal Peptide), ELP (Elastin Like Peptide), and others, and highlights both FDA-approved products and candidates currently in different stages of clinical development. In the meantime, special attention has been paid to albumin-binding domains and albumin-binding domain antibody (AlbudAb), which increases the half-life of biological drugs by binding to albumin, as the most abundant and stable protein in the body.

Rotaviruses (RV) are a major cause of severe childhood diarrhoea, particularly in developing nations, necessitating stable vaccines. Therefore, the presented preliminary study aimed to assess the impact of altered physicochemical properties on the structural stability of recombinant rotavirus capsid protein VP6 (RV-VP6). The expression system used in this study was designed by genetically engineering the RV-VP6 into E. coli (NiCo21(DE3))-pET28a host-vector system and purified using liquid chromatography. The purified RV-VP6 homology detection and structure prediction were conducted using LC-MS and HHpred computational analysis, which indicated a 100% probability of 1QHD_A Viral Capsid VP6 (1.95 Å), representing the crystal structure of VP6. The secondary and tertiary structural stability of RV-VP6 was evaluated in altered pH and Ca²⁺ concentrations using far UV-CD and intrinsic tryptophan fluorescence spectroscopy, respectively. The computational analysis of the far-UV CD spectra revealed a significant increase in the composition of α-helices and β-sheets in altered pH and Ca²⁺ environments compared to the denatured protein (p < 0.0001). Intrinsic fluorescence analysis of RV-VP6 at pH 7 yielded an emission λmax of 339 nm, which shifted to 342 nm at pH 5. In 1 mM Ca²⁺, a λmax of 340 nm was observed, with an increase in intensity in 10 mM Ca²⁺, accompanied by a slight blue shift to 338 nm. Investigation of RV-VP6 under thermal stress yielded unfolding concomitant with aggregation, rendering the process irreversible and nullifying analysis using equilibrium thermodynamics. These findings form the preliminary basis for our future evaluation of manufacturing stable and enhanced RV-VP6 vaccines through the downstream process control of (1) pH, which alters the charge distribution on the surface of the protein, leading to conformational changes, and (2) Ca²⁺ ions, which interact with specific amino acid residues in the protein, thereby affecting its structure and function.